Absorbance and Fluorescence Detection for HPLC Separations

One popular HPLC detector takes advantage of an analyte’s UV/Vis absorption spectrum. These detectors range from simple designs, in which the analytical wavelength is selected using appropriate filters, to a modified spectrophotometer in which the sample compartment includes a flow cell. The illustration here shows the design of a typical flow cell for a detector using a diode array spectrometer. The flow cell has a volume of 1–10 μL and a path length of 0.2–1 cm.

Figure12.46

The resulting chromatogram is a plot of absorbance as a function of elution time.  The example shown here is for an HPLC separation of a mixture of flavonoids with detection at 360 nm and at 260 nm. The choice of wavelength affects each analyte’s signal. By carefully choosing the wavelength, we can enhance the signal for the analytes of greatest interest. (The data for this figure was provided by Chistopher Schardon, Kyle Meinhardt, and Michelle Bushey, Department of Chemistry, Trinty University.)

Figure12.47

If an analyte is fluorescent, we can place the flow cell in a spectrofluorimeter. As shown here for the determination of of riboflavin in urine, a fluorescence detector provides additional selectivity because only a few of a sample’s components are fluorescent.

Figure12.48Fluorescence measurements were made using an excitation wavelength of 340 nm with detection at 450 nm. The peak corresponding to riboflavin is marked with a red asterisk (*). The inset shows the same chromatogram when using a less-selective UV/Vis detector at a wavelength of 450 nm.

Data provided by Jason Schultz, Jonna Berry, Kaelene Lundstrom, and Dwight Stoll, Department of Chemistry, Gustavus Adolphus College.

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