Spectrophotometry is a highly regarded analytical technique that has found widespread use in the fields of chemistry, biochemistry, and physics research. Among the various types of spectrophotometry, fluorescence spectroscopy stands out as a particularly powerful tool for measuring the fluorescence emissions of a sample. This non-destructive technique has found extensive application in diverse fields such as biochemistry, environmental monitoring, and pharmaceutical analysis.

By exploring the intricacies of fluorescence spectroscopy in this article, we hope to provide a comprehensive understanding of this analytical technique and its potential applications. Whether you are a seasoned researcher or a curious student, this article will serve as a valuable resource for gaining insight into the fascinating world of fluorescence spectroscopy.

1. Introduction to Fluorescence Measurement

Fluorescence measurement is a type of spectrophotometry that measures the light emitted by a sample when it is excited by specific wavelengths of light. This technique is based on the principle that when a molecule absorbs light of a specific wavelength, it becomes excited and emits light at a different wavelength. The emitted light is referred to as fluorescence and can be measured to determine the concentration of the substance in the sample.

Spectrometers are essential in fluorescence measurement since they allow scientists to precisely measure the intensity and wavelength of the fluorescent light emitted by a sample. This information provides insights into the sample's chemical and physical properties, such as the presence and concentration of specific compounds.

2. Principles of Fluorescence Spectroscopy

• Fluorescence Process: When a molecule absorbs light in the visible or ultraviolet region of the spectrum, the electrons in the molecule are excited from the ground state to the excited state. These electrons tend to return to the ground state, releasing absorbed energy as heat and radiation in the visible or near infrared spectrum. The emitted light is called fluorescence (or phosphorescence if the excited state is a triplet state).
• Stokes Shift: The Stokes shift is the difference between the wavelength of the excitation light and the wavelength of the emitted fluorescence light. This shift occurs because some of the energy absorbed by the molecule during excitation is lost through non-radiative processes, such as heat. As a result, the emitted fluorescence has a longer wavelength than the excitation light.
• Quantum Yield and Fluorescence Lifetime: The quantum yield is a measure of the efficiency of the fluorescence process and is defined as the ratio of the number of photons emitted to the number of photons absorbed. The fluorescence lifetime is the average time that a molecule spends in its excited state before returning to its ground state. These two parameters can be used to determine the concentration of a substance in a sample.

3. Overview of Ocean Insight Fluorescence Measurement Packages

As the demand for more accurate and reliable fluorescence measurements continues to increase, the development of more advanced and sophisticated spectrometers is expected. The Ocean Optics (Ocean Insight) Fluorescence Measurement Packages such as BUNDLE-QEPRO-FL models are an excellent example of a spectrometer-based system that is easy to use and provides accurate and reliable results. These Fluorescence Measurement Packages provide a comprehensive and reliable solution for fluorescence measurement using spectrometers.

Figure 1. The Fluorescence Measurement Packages from Ocean Insight

The Fluorescence Measurement Packages Features and Capabilities

• Spectrometer: QE Pro-FL and Ocean SR6 series – a high-performance spectrometer that covers a broad range of spectral ranges from ultraviolet to near-infrared. It features industry-leading optical resolution, low stray light, and high-sensitivity performance, making it ideal for fluorescence measurements.
• Convenient bundle -- each package comprises everything you need for cuvette-based fluorescence measurements
• Powerful excitation source -- 365 nm LED couples to optical fiber with great efficiency
• Easy to use -- Plug and play via the micro USB connection
• User interchangeable slit — allows you to vary the resolution and throughput of the spectrometer on demand

4. Best practices for using Ocean Insight Fluorescence Measurement Packages

• Sample Preparation: The sample should be homogenous, free from contaminants, and properly diluted to avoid quenching effects. For solid samples, proper grinding and dispersing can help ensure uniformity.
• Setting Up the System: Proper setup and configuration of the spectrometer are essential for obtaining accurate and reliable measurements. The hardware and software components of the system should be installed and configured according to the manufacturer's instructions.

Figure 2. Complete system for measuring Fluorescence

• Calibration and Validation: Before taking fluorescence measurements, it's essential to calibrate and validate the instrument. This involves using known reference standards and control samples to establish a linear response curve and ensure the accuracy of the measurements.
• Performing Measurements: During measurements, it's important to maintain consistent measurement conditions, such as temperature and light conditions, to minimize variability. Multiple measurements should be taken to increase confidence in the results, and any outliers or inconsistent results should be investigated.
• Analyzing Data: After measurements are taken, the data should be analyzed using the OceanView software that comes with the package. The results should be compared to reference standards and control samples to ensure accuracy, and any abnormalities or unexpected results should be investigated and explained.