Although scientists have relied solely on LC-MS methods in recent years, HPLC-fluorescence detection is now designated as the official analysis method for many components across various industries. In particular, it is used for analyses that demand high sensitivity and high selectivity, especially when the analyte has little or no UV absorbance.

Specifically, fluorescence is widely used in the food, environmental, and pharmaceutical fields, especially with samples containing high levels of impurities. One example is the pharmaceutical industry, where fluorescence detection combined with either HPLC or high-performance thin-layer chromatography (HPTLC) has been used for pharmaceutical quality control in hospital chemotherapy production units.¹

HPLC-fluorescence detection has a number of important benefits, including high sensitivity, high selectivity, and repeatability. The most advanced fluorescence detectors feature a temperature-controlled cell to ensure stable analysis even if the ambient temperature fluctuates. These detectors also provide high levels of sensitivity and validation to support functions in a wide range of applications from conventional to ultra-fast LC analysis.

Fluorescence detection: State of the art

Scientists seeking the most reliable and efficient HPLC-fluorescence should consider the following factors when selecting instrumentation.

High sensitivity

With a water Raman S/N ratio of at least 2,000, the most advanced fluorescence detectors are powerful tools for tests demanding analysis of trace-level components while retaining the acquisition speeds necessary for ultrafast analysis. Because fast response is necessary to follow the sharp peaks in ultra-fast analysis, some of these instruments can provide 10 ms response. The quick response time permits ultra-fast LC without loss of separation.

Additionally, simultaneous testing of multiple components requires detection at optimal wavelengths. These detectors allow ultra-fast, highly sensitive, multicomponent analysis using wavelength switching via a time program.

Temperature control

As a general rule, fluorescence intensity drops as the temperature rises, because the molecular collisions increase in frequency with increases in the temperature. Therefore, molecules lose their potential energy. In other words, a fluctuation of the ambient (detection) temperature changes the fluorescence intensity of some compounds and this negatively influences the accuracy of analysis. However, more advanced instruments have a temperature-controlled cell as a standard feature, ensuring the high reliability of analysis that is not affected by temperature.

Figure 1. Effect of cell temperature on peak intensity.

Figure 1 shows a comparison of the peak intensities at cell temperatures of 25°C and 30°C. The comparison of two chromatograms at cell temperatures of 25°C and 30°C reveals a decrease in peak intensity of more than 10 percent for every compound at the higher cell temperature. By maintaining a constant cell temperature, peak intensity, and detection, sensitivity will not be compromised if the room temperature changes during the sequence run.

Figure 1. Effect of cell temperature on peak intensity.

Modern detectors should include a temperature-controlled cell with a cooling function. This maintains a constant detector cell temperature, even if the testing environment’s room temperature fluctuates, so it ensures reproducibility without any drop in sensitivity.

Arc lamps vs. flash lamps

There are a number of differences between xenon arc lamps and flash lamps in fluorescence detectors. For example, the life of xenon arc lamps can last up to 2,000 hours, which can be up to four times longer than flash lamps. This significantly reduces running costs and downtime due to maintenance.

However, it should be noted that the life of either type of lamp is impacted by the way the lamp is run, so more flashes at a lower energy (joules) or less flashes at a higher energy may result in the same lifespan. The typical lifetime also depends on the starting voltage, peak current, length of discharge, and fill pressure. While many lamps are designed to last one million to 10 million flashes under “normal” conditions, changing the energy per flash can shorten those numbers significantly.

Another consideration is that arc lamps change output wavelengths, depending on how much energy is used. At lower flash energy, users will get more ultraviolet light in the 300 to 400 nm range. At higher flash energy, the energy shifts to visible and infrared light in the 700 to 1,000 nm range. Top-of-the-line detectors have a power supply that provides consistent energy flash-to-flash so that the energy expended per flash does not need to be monitored as closely.

This allows flash lamps to be tuned to the sensitivity requirements of the analysis. However, when used at their most sensitive and highest flash frequency, the lifetime is normally below that of a xenon arc lamp, making the cost of ownership higher.

Analyzing food additives

Figure 2. Chromatogram of TBHQ (0.001 mg/L, 5 µL injected).

Tert-Butylhydroquinone (TBHQ) is one of the phenolic antioxidants, and its use as a food additive is permitted in some countries. Typically, HPLC with UV detection is employed for analyzing phenolic antioxidants; however, some of these substances, including TBHQ , can be detected with a fluorescence detector. Figure 2 shows an example of TBHQ high-sensitivity analysis using an advanced fluorescence detector. Here, 5 µL of a TBHQ standard solution (0.001 mg/L) was injected, clearly demonstrating that 5 mg of TBHQ can be detected.

Environmental testing

The recent oil spill in the Gulf of Mexico created concerns over the safety of seafood that could be contaminated with polycyclic aromatic hydrocarbons (PAH) from crude oil. PAH is one group of wide-ranging environmental pollutants, some of which the U.S. Environmental Protection Agency has identified as carcinogenic, toxic, and mutagenic. The EPA has designated 16 PAH compounds as “priority pollutants” that are subject to restriction.

Some Gulf jurisdictions started screening seafood for at least 12 PAH compounds. The traditional method for analyzing PAH is GC-MS, which can take upwards of 60 minutes per analysis. However, because many PAH compounds are fluorescent, they can be detected with high selectivity and high sensitivity using a fluorescence detector.

Figure 3. Separation of polycyclic aromatic hydrocarbons at 20 ppb level (top); Samples of tuna and oyster vs. the same samples spiked with PAHs at ppb level.

In one analysis, researchers performed rapid analysis of PAH in tuna and oysters using UHPLC equipped with a sub 2-micron PAH column and fluorescence detector. The 100 Hz sampling rate permitted ultra-fast LC with no loss of separation. Furthermore, this method provided accurate determination of 15 PAH compounds with optimum wavelengths in less than four minutes. (See Figure 3.)

Pharmaceutical analysis

Tocopherols (Vitamin E) consist of a class of nutrients that are not only used as food additives such as antioxidants and nutritional supplements, but are also included in natural ingredients. Tocopherols are known to exist in various forms, including the ß-, ?-, and d-isomers, as well as a-tocopherol.

Figure 4. Chromatograms of standard mixture of tocopherols (2 mg/L each).

HPLC analysis of tocopherol isomers is typically conducted using normal phase chromatography combined with fluorescence detection. In this example, ultra-fast analysis was conducted using a highspeed, high-resolution analytical column (particle diameter 2.2 µm) together with a fluorescence detector.

Figure 4 shows an example of analysis of a standard solution of four tocopherols (2 mg/L each) using a conventional column (particle diameter 5 µm) and the highspeed column. The high-speed column shortened the analysis time to less than one-fourth of the original time.

Conclusion

Fluorescence detectors are used in a wide range of fields and are indispensible for their high sensitivity and selectivity. With the capacity to identify and quantify low concentrations of target compounds, they are the right complements to LC in applications requiring efficient and reliable analysis.