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About Ocean Optics
Fluorescence spectroscopy analysis is an excellent tool for analytical science and investigational research applications. It finds applications in chemical, biochemical, pharmaceutical and medical sectors as well as in fluorescent labeling, mineralogy, sensors and fluorescent applications. It is also used for identifying proteins, oils, dyes and organic compounds and used for laser- induced chlorophyll fluorescence and environmental monitoring for crop yield assessments.
This spectroscopy type is targeted at a sample’s vibrational states. Certain substances can be excited to a higher electronic phase by using a specific frequency. A particular excitation may deliver a fluorescence or emission peak.
A number of options are offered by Ocean Optics of ready-to-use fluorometry systems having a number of resolutions, time-gating options and off-the-shelf configurations. LVF high pass and low pass filters, cuvette holders, a fiber optic scanning monochromator and a range of excitation sources are available. The fluorescence spectrometers can sense fluorophores in powders and liquids as well as from surfaces. Anthrax can be detected using the USB2000-FLG in their EDS2000 System and to detect fluorescence in coral, fruit, and other flora and fauna.
The steps followed for the experiment are:
- Standard stock solutions of quinine sulfate solution in sulfuric acid and methanol were prepared with approximate concentrations: 1, 20, 40, 60, 80, and 100 ug/mL.
- Smaller concentrations of quinine sulfate stock solutions of 0.50, 0.25, 0.06, 0.03, 0.01, and 0.00 were also created.
- These changing concentrations of quinine sulfate standard stock solutions were tested for fluorescence using CVFL-Q-10 quartz cuvettes in a CUV-ALL 4-way cuvette holder.
- These measurements were done using a QE65000 spectrometer (grating #HCI, 200 pm slit, range 349.2 nm — 1143.5 nm, optical resolution 6.4 nm (FWHM), PX-2 light source, HR4-BREAKOUT Breakout Box, MonoScan2000 scanning monochromator, and SpectraSuite software.
- Three QPI000-2-UV-VlS fibers were utilized for connecting the PX-2 light source to the MonoScan2000, the MonoScan2000 to the CU V-ALL cuvette holder, and the CU V-ALL cuvette holder to the QE65000 spectrometer. Fibers were attached to the cuvette holder at 90 degrees as shown in Figure 1.
Figure 1. Equipment set up for fluorescence.
- Fibers were taped down to bring down attenuation from movement.
- The PX-2 light source was warmed-up for 15 minutes before measurements.
- A new dark measurement was stored and subtracted between every change in sample and/or every 15 minutes to reduce error and drift.
- Spectra were recorded between 375-600 nm.
- The modes used for recoding measurements include both scope mode and relative irradiance mode.
- Scope mode data is unprocessed with the instrument response function not factored out.
- Relative irradiance measurements were performed using the same equipment, but with the addition of a LS-1 tungsten halogen light source that was used as a black body reference with known color temperature.
Emission, or fluorescence, peak spectra were collected from various concentrations of quinine sulfate stock solutions in both scope and relative irradiance modes. Replicates measurements were taken to create calibration curves. Peak locations varied slightly (more so in Scope Mode) from the reported 450 nm maximum fluorescence peak for quinine sulfate. In scope mode concentrations from 20-100% solutions peaked at 457.84 nm.
The one percent solution replicates were the most variable in the measured peak location. The peak height locations for these replicate measurements were averaged and rounded to the nearest nanometer for reporting on the calibration curve. The measured peak for fluorescence in relative irradiance was found to be 449.11 nm for all concentrations. Calibration curves were created for two different concentration ranges. See Figures 2 and 3.
Figure 2. a. Calibration curve of quinine sulfate from fluorescence peak points in scope data at concentrations from 1- 100 ugfmL. b. Calibration curve of quinine sulfate from fluorescence peak points at 449.11 nm from relative irradiance data. This graph illustrates the linear range at much smaller concentrations. c. Emission spectra of quinine sulfate solutions.
The most linear part of the calibration curve is in the small concentrations range under I ug/mL (or I ppm or 1000 ppb). As anticipated, concentrations of fluorophore, due to the innerfilter effect, the excitation drops off after reaching a maximum intensity. There was a good linearity on the calibration curve for the smaller concentrations of quinine sulfate. The R2 value for linearity of the curve was 0.998 using the relative irradiance data.
Relative irradiance data proved to be more consistent over all and was much closer to the reported fluorescence peak locations of quinine sulfate in published literature. A quinine calibration curve is apt for checking quinine concentrations in liquids for quantitative analysis.
The QE65000 Scientific-grade Spectrometer is a sensitive system great for low-light level applications such as fluorescence. Since the QE65000 can achieve up to 90% quantum efficiency with high signal-to-noise and rapid signal processing speed, this would be the preferred spectrometer for fluorescence applications.
About Ocean Optics
Ocean Optics is a diversified photonics technology firm and a global leader in optical sensing. With full-service locations in the United States, Europe and Asia, Ocean Optics serve a wide range of markets, including process control, consumer electronics and medical diagnostics.
This information has been sourced, reviewed and adapted from materials provided by Ocean Optics.
For more information on this source, please visit Ocean Optics.