A Spectroscopy Guide for PAT

Figure 3: High Speed Chromatogram. Click for larger image.

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.

Fluorescence (FL)

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.

Simple Fluorescence (On-line)

Where applicable, these monitors (grating or filter) can give molecular information with little interference from the solvent. Since the emission spectra of a fluorescing molecule has a specific spectrum and is quite sensitive (low analyte concentration), a number of reactions may be followed in detail within complex systems.

HPLC

Highly sensitive, fluorescence monitors may be scanning or simple filter types. These are, simply, smaller samplesized versions of an on-line monitor. With the small sample size used in analytical chromatography, fluorescence detectors may be placed in tandem with UV detectors. Typical analytes include salicylic acid and ergot alkaloids; both present in small to minute levels. Alkaloids are potent and may be present in low levels, while salicylic acid is an impurity in aspirin and must be quantified in all dosage forms containing aspirin (production and stability samples) down to parts per million levels.

Light-Induced Fluorescence (LIF)

Initially the “L” in LIF stood for laser, but lately, laser diodes have become popular as light sources due to their high-intensity, monochromatic light. In cases where lower concentrations of drug substance are present (less than 1% relative), LIF outperforms NIR and Raman. One such case would be in blend uniformity of a mixture, prior to either compression or granulation.

Figure 4: LIF Monitor on  Tablet Press. Click for larger image.

Figure 4 shows an LIF probe attached directly to a tableting machine, monitoring 100% of the tablets produced. While LIF has many of the limitations of normal fluorescence, the higher light flux may induce fluorescence in materials not normally considered candidates for fluorescence analysis. Other than NIR, LIF is one of the few spectral techniques usable on powder mixtures.

Laser-Induced Breakdown Spectroscopy (LIBS)

LIBS has been around for years, but isn’t often mentioned alongside “common” PAT tools. It consists of a finely focused laser that strikes, for example, a tablet core or coated tablet, causing vaporization at the point of contact. The light emitted is studied for metals and a number of other elements, such as nitrogen and halides. The time at which the light is measured gives elemental or molecular information. The point of laser contact is hit with subsequent pulses, causing the beam to, in essence, drill a small hole into the tablet, extruded material, or whatever the target. The emission spectra gathered provide a depth-profile of various elements at that point. The three dimensional pattern formed is shown in Figure 5.

Figure 5: LIBS Profile on Tablet. Click for larger image.

By repeating the operation at adjacent points on the sample, the analyst generates a three-dimensional picture of the distribution of, for instance, the magnesium stearate in the tablet or titanium dioxide in the coating. This is obviously a destructive technique and not very fast, but is an excellent tool to show the homogeneity of both a tablet matrix and its coating. Root cause analysis would be one obvious use for LIBS.