Zechariah Kitzhaber

The first installment of this three-part series provided a procedure for quickly recording acceptable fluorescence spectra with classic commercial spectrofluorometers for most samples in common 1-cm pathlength cuvettes. Part 2 described how to correct for instrument response so data can be compared properly between laboratories. This part describes sample-specific concerns that could require modifications of our basic procedure to prevent our data from being inconsistent.

In parts 1 and 2 of this article (1,2), we described a simple procedure to select conditions for spectrofluorometry in 1-cm cuvette samples, and how to determine and correct data for instrument artifacts. These procedures are adequate for comparing spectrofluorometry between laboratories, as well as for producing data that is consistent within the same laboratory over time or with different instruments, in most cases. The remaining complications of conventional spectrofluorometry can be ascribed to sample-specific concerns that cause the sample fluorescence itself to be inconsistent. Part 3 of this series discusses reasons that samples might give inconsistent results even when you have chosen what appear to be appropriate instrument settings, and considered every potential artifact and defect of your measurement system. We primarily focus on measurements with continuous (not pulsed) light sources.

Molecules that emit light can be sensitive to their environments, adversely affecting interlaboratory comparisons if environmental factors are not understood and controlled. Conversely, many chemical or physical phenomena that cause irreproducibility in spectrofluorometry are also potential targets for fluorescence sensing applications, so it is advisable for an analyst to be aware of the mechanisms of these effects. We consider the most obvious elements of sample condition first:

Solvents greatly exceed solutes in concentration, and the effect of a solvent on spectrofluorometry can therefore be substantial. Solvents (even “spectroscopic grade” solvents) may have fluorescent impurities (3); these could have come in the fresh solvent, or have been introduced through poor laboratory technique, detergents, or contaminated glassware (4). Some solvents may also have non-fluorescent stabilizers intentionally added (5,6). For example, diethyl ether, an aprotic solvent, is sometimes stabilized with ethanol, a protic solvent. Also, fresh solvents, left exposed to air, will dissolve atmospheric gases including moisture. Traces of water in a non-aqueous solvent can have significant effects on fluorescence compared to a scrupulously dry non-aqueous solvent (for example, chlorophyll fluoresces more strongly in protic solvents). Traces of acetone sometimes found in solvents are also known to affect fluorescence (7).

Even clean solvents interact with solute molecules through their dipole moments and polarizabilities in ways that cause shifts in spectra, changes in apparent intensity, changes in the quantum yield of luminescence, and changes in bandwidths (8). These effects are fairly common to all emit- ting molecules. In some cases, however, solvents can have additional effects on the equilibrium distribution of molecular conformations, the protonation state of molecules, or the ordering of excited states (9). Behera, Park, and Gierschner have recently provided a systematic organization of the various types of dual-emitting samples (10), and we can anticipate specific solvent effects for nearly all these cases. It should be clear to the reader that the choice of solvent is one of those measurement conditions that must be recorded and reported to have any hope of repeatability.

We refer to the molecule on which we are performing spectrofluorometry as the “target compound,” to distinguish it from other fluorescent species in the sample. The purity of target compounds used for spectrofluorometry is a potential source for concern when comparing results. The lower the quantum efficiency of the target compound, the more concerning any potential strongly fluorescent contaminant will be (11).

If the target compound is purchased, the purity is usually specified as a minimum percentage by weight, giving an upper limit to the amount of all impurities. If the target compound is synthesized locally, quantitative nuclear magnetic resonance (NMR) appears to hold promise for detecting all impurities (including water), as long as they are amenable to NMR study (12). Fluorescent impurities (such as chemical relatives of the target compound that have not been adequately separated) have historically been detected by some form of chromatography (for example, from simple thin layer chromatography to high performance liquid chromatography [HPLC]) using fluorescence detection (including visually inspecting for fluorescent bands).

Target compounds stored in solution in clear bottles or for long periods of time are likely to be thermally degraded over time, leading to more contaminants than were originally present. For instance, many organic dyes degrade when exposed to oxygen in solution, so even a dark bottle is not sufficient to avoid making fresh solutions for calibrations. If you want to keep your target compound as nearly pure as possible, keep it in the solid form, in its original container, in the dark, and avoid heat. If you refrigerate it, wait for it to warm before opening to avoid condensation.

Increasing temperature ordinarily decreases the yield of luminescence in the absence of other effects by increasing non-radiative decay processes for isolated molecules in solution. This change is typically in the range of 1% per 

C, although some systems can show up to 5% per degree (4) There can be exceptions with the opposite response to temperature, at least over limited temperature ranges, because of changes in thermal equilibrium distributions of conformations, association equilibria or thermally accessible excited states, and other reasons.

Pressure effects are usually much less pronounced than temperature effects under near-ambient conditions. However, one significant effect of pressure is to change the equilibrium concentration of dissolved atmospheric gases (especially oxygen) in the solution. This effect has been used as the basis for fluorescence-based (actually phosphorescence-based) oxygen and pressure sensors (13).

Concentration of the target compound affects spectrofluorometry in three important ways. The first of these is through the inner-filter effect (18). The inner-filter effect can itself be divided into primary and secondary types, the former caused by absorption of the excitation beam by the sample, and the latter caused by re-absorption of the fluorescence before it emerges from the sample. If the procedure described in Part I is followed, these effects should be real, but small and correctable (8).

The second sample concentration effect is through the possible formation of dimers and aggregates (14) in the ground state, excited state, or both. Carboxylic acids (15) and laser dyes (16) are well known to exhibit dimerization in solution in their ground states, whereas pyrene is the classic example of a molecule that can form excited dimers (also known as excimers) (17). Dimerization can result in a change in the absorption spectrum, a change in the fluorescence spectrum, concentration quenching, or a change in the fluorescence quantum yield of molecules in solution.

The third effect of sample concentration is through excited state energy transfer between different identical molecules that can depolarize fluorescence (14), a factor that is only important in situations where the fluorescence is not already depolarized for other reasons, or where it has not been corrected for in instrumentation (see Part 2).

We called these first five sources of irreproducibility in spectrofluorometry obvious because solvents, sample purity, temperature, pressure, and concentration are the state variables that many of us learn about in introductory courses in chemistry. However, there are some less obvious sources of irreproducibility that we’d like to mention. These include polarization of fluorescence, long-lived phosphorescence, quenching, photochemical decay, and dissolved ion concentration.

We mentioned in Part 2 that samples may exhibit polarized fluorescence, and, if they do, this needs to be accommodated in the instrument with polarizers placed appropriately. We did not, at that point, explain why molecules might exhibit polarized fluorescence.

Because of the symmetry of the dipole moment operator that enables most light absorption and emission, both excitation and emission processes of target compounds are guaranteed to be inherently polarized along one or two directions in the molecule. The only exceptions are optical transitions involving molecular species of very high symmetry—molecules of exactly tetrahedral, octahedral, or even higher symmetry. In a 90-degree instrument configuration as discussed above, even unpolarized excitation can result in partially polarized emission (8,18). Macromolecules like proteins, or small molecules bound to macromolecules and macromolecular assemblies like micelles or cyclodextrins, can rotate in solution so slowly that polarized emission is preserved. In this case, the presence of certain surfactants or macromolecules in solution can change the polarization state of emission even for small molecules with normal fluorescence lifetimes. There are also cases where the fluorescence lifetime of a small molecule is so short that polarization of emission is still detectable, even without viscous solvents or association with a macromolecule (19). Any residual polarization of the emission from a target compound can change the relative intensity of observed fluorescence, and it can vary with excitation wavelength because different optical transitions can be excited across the excitation wavelength range. Part 2 provides the means of correcting for this potential problem.

Phosphorescence is distinguished experimentally from fluorescence by the lifetime of its exponential decay following a pulse of light, typically being orders of magnitude slower than fluorescence. In rare cases, phosphorescence can have lifetimes on the order of seconds. This gives rise to two important concerns.

First, when working with long-lived phosphorescent molecules, emission intensity does not change promptly when the excitation is turned on or shuttered, but requires multiple half-lives to reach a steady-state intensity. An excitation spectrum can be “blurred” if it is scanned faster than the sample phosphorescence equilibrates. Likewise, repeated excitation scans may include moments where the excitation wavelength changes at the maximum slew rate of the excitation monochromator to start the next run, and the start of a subsequent spectral measurement could be affected by the sample’s “memory” of recent excitation conditions. For the same reason, emission spectra can show artifacts at the start of a new measurement because they begin too quickly after the excitation is unblocked or the wavelength of excitation is reset to the measurement condition. Fortunately, only a fraction of luminescent molecules possess decay rates long enough (>1/5th of the dwell or integration time) for long-lived phosphorescence to cause a problem like this.

However, nearly all phosphorescent molecules are subject to quenching of their emission. This is because even short-lived phosphorescence is orders of magnitude longer than most fluorescence, giving ample time for molecules in solution to find and interact with other molecules that can quench their emission. Just as important, there is a very common molecule that is an excellent quenching agent for phosphorescence: molecular oxygen.

Most of the emitting excited states of phosphorescent molecules have unpaired electron spins. Molecular oxygen has a number of low-lying excited states that could accept energy from an excited molecule, and also has unpaired electron spins. Because of the relatively long-range magnetic coupling between the unpaired spins of molecular oxygen and those of other phosphorescent molecules, a small amount of dissolved oxygen can be very effective at quenching the energy from excited phosphorescent molecules before they can emit light (11). Because the analyst has probably not intentionally introduced oxygen to their spectrofluorometer sample, the oxygen concentration and thus the phosphorescence intensity may vary significantly between samples—or even for exactly the same sample from the moment it is freshly made to when it equilibrates with the atmosphere.

Quenching by molecular oxygen is a very important type of quenching for phosphorescent molecules. However, all excited molecules can show quenching phenomena under the right conditions. The presence of contaminants in the solvent, or in an impure target compound, can quench the fluorescence of the target compound. The presence of dimers of the target compound can also quench fluorescence from isolated molecules.

Photochemistry is another of the possible non-radiative decay pathways for excited molecules, and it can result in conversion of our target compound to other chemical structures or con- formations. In rare cases, photochemical reactions may be reversible (such as, for example, spiropyrans [20]), but usually not. The products of photochemical decay may be colorless or strongly colored; they may be fluorescent themselves, or not fluorescent. They may in some cases act as quenching agents when they build up.

Photochemistry is nearly always more important in fluid solutions than in solids; this is another reason target compounds are best stored in a solid form if not immediately being used. Multiple photochemical products of a target compound may be possible, and the yield of each may vary differently with wavelength (21). More photochemical pathways will probably be opened with high energy (such as UV) than with low energy excitation, which will not surprise the sunburned among us.

However, one absolute requirement for photochemistry is that photons be involved. Photochemistry cannot happen in the absence of light absorption, so the traditional cure for photochemistry that might occur during a study is to keep the target compound in the dark, or only illuminate it with long wavelengths of light, both before spectrofluorometry and whenever possible during a study.

Some molecules are particularly susceptible to changes in their absorption and fluorescence spectra due to changes in surfactant, H

 and other ion concentrations, or other trace molecules. In addition to possible polarization effects, surfactant micelles can form local environments and local concentrations of a target compound, and give rise to dimerization or other effects (22). Target compounds with acidic or basic groups may show substantial changes in spectroscopy and quantum yield, depending on their protonation state. Complexation with metal ions can also produce changes in fluorescence.

Procedure: Prepare → Evaluate → Measure 

When publishing or transmitting your data to others, it is important to specify as much of what you have done as possible to enable others to reproduce your work.

Begin with a clean, fluorescence-free cuvette with low absorbance. When buying cuvettes, manufacturers may describe the materials from which their cuvettes are made in detail (by specific composition), and this is useful information to know. Quartz or fused silica are often the materials of choice for fluorescence-free cuvettes. However, after settling on high quality cuvettes, you will need to confirm the lack of measurable fluorescence in your instrument, and will need to keep them clean to maintain that performance. Manufacturers of cuvettes and spectrofluorometers can provide one or more protocols for cleaning cuvettes to avoid contaminants. Protocols may include, among other steps, soaking in nitric acid, rinsing with deionized water, or cleaning with a detergent such as Alconox. HNO

protocols are particularly effective at removing organic contaminants with little residue. Chromic acid solutions are often used for cleaning laboratory glassware, but they absorb UV, and so have never been recommended for cleaning optical materials. Phosphoric acid, HF, and strong bases are not suited to cleaning optical materials because they etch glass and silica. Also, be aware that quartz or fused silica cuvettes are not usually intended for high temperature or ultrasonic processing.

Ensure you have an appropriate, fluorescence-free solvent of high purity. An appropriate solvent would be one whose evaporation rate is low or can be controlled, that dissolves the sample well, and whose likely effect on the solute is understood. Regardless of the purity of the solvent, or whether it says “spectroscopic” or “HPLC” grade on its label, a blank cuvette of the solvent should be run to confirm low absorbance across the spectral window of interest and low fluorescence. The blank solvent spectra can be used as a background for the eventual measurement of the solute. If the purity of the solvent is of concern, it has been observed that filtering a solvent through activated carbon followed by distillation is effective for removing many organic contaminants. Molecular sieves can reduce moisture levels to sub-10 ppm levels (23); other more active techniques can reduce moisture even lower. Electrochemistry texts provide excellent procedures for removing water from solvents by vacuum line techniques if this is an issue (24). Note that benzene and related compounds may sometimes found as contaminants in several solvents otherwise suitable for UV studies (25), so it is a good idea to check for any deep-UV-excited fluorescence in a solvent.

The solvent should have a well-defined concentration of known ions, and a known pH if using aqueous solutions with solutes that have acid-base properties. Most fresh solvents have been assayed for their major metals and ionic species. Samples taken from nature may require clean-up steps to remove unwanted species, or steps to ensure the pH is maintained at a desired level without introducing new chemical species to the solution.

The target compound you intend to study should ideally be of a known, high purity. One common approach to checking purity is to see if the fluorescence excitation spectrum of sample is similar to the absorption spectrum of the target compound (11), and that the fluorescence spectrum itself is independent of the excitation wavelength. These procedures are sometimes misleading and are not very sensitive to low levels of contaminants, so if you’re not sure, high-performance liquid chromatography (HPLC) with fluorescence detection is a very effective way to test for fluorescent impurities.

Long-lived phosphorescence of the sample under these measurement conditions can either be an inherent property of the sample, or evidence of an impurity (such as a free ligand in the solution of a coordination complex). We assume here that the time between measurements in the measurement protocol is longer than ~200 ms, because of the combination of dwell time at each point and the time required to change wavelengths. For the purpose of detecting luminescence with a lifetime long enough to require slowing data acquisition further, detection can often be achieved with the naked eye, as long as the emission is visible. One can hold the emission filter from the spectrometer in front of the unaided eye, and see the emission coming from the sample, while using an opaque card to the block the excitation beam. If there is a discernible lag between blocking the beam and the end of emission to the naked eye, then there is likely a long-lived phosphorescent component to the emission. If you worry your eyes are fooling you, many of us have an optical detector with ~30 ms time resolution in our pocket (our cell phone cameras), and, if the emission is bright enough, a cell phone video can be used to confirm the observation. If still in doubt, the sample can be subjected to a fluorescence lifetime measurement, which usually requires another instrument. If a long-lived emission is present, an increased delay between measurements may be needed to allow the emission to equilibrate. In the case that there is a phosphorescent contaminant in the sample that cannot be removed, and when the target compound itself is not phosphorescent, it may be possible to eliminate the contaminant’s phosphorescence, and retain the desired target compound fluorescence by saturating the sample with oxygen and sealing it. This will not, however, be helpful if the target compound has.

To determine whether the emission of the target compound is sensitive to oxygen at the selected concentration, we can follow a published protocol (26). We begin by setting up the fluorimeter for a measurement with your planned spectral bandwidth (SBW) and other instrument parameters, only without the sample in place. Set the excitation and emission wavelengths to their optimum positions based on preliminary studies, and prepare to record a time-scan using constant excitation and emission wavelengths. While the instrument is sitting ready, place a septum over your cuvette containing about 3 mL of your experimental solution (a typical 1-cm cuvette sample). Place a syringe needle through the septum into the air space above the sample as a vent, and another that passes to the bottom of the sample. This latter syringe should then be connected to a low flow rate of dry nitrogen gas so that bubbles of N

 are observable in the sample. Continue purging with N

 for 7 min to degas oxygen from the sample, and then remove both needles. Immediately transfer the sample to the fluorimeter and measure the fluorescence intensity for a period of time (about a minute) to ensure it is stable, or at least changing very slowly and consistently. Leaving the cuvette in the instrument, place the syringe needles back into the cuvette and bubble fresh air into the solution using a bulb until enough has been added to displace the gas above the sample, taking care not to suck the sample back into the bulb. Remove the needles and observe the intensity of the sample relative to the purged sample. Small changes can be ascribed to changes in sample position, but large changes suggest significant oxygen sensitivity. The changes in intensity for four sensitive compounds reported by Pagano (26) ranged from a factor of 3.6 to a factor of 21.8. If your sample shows oxygen sensitivity, it should be prepared for study by purging with N

 and leaving it sealed during analysis. If you need to purge the sample, be aware that a correction to the concentration because of solvent evaporation will be required, if your solvent is at all volatile.

If your target compound shows no immediate loss of signal from introduction of oxygen in the preceding test, continue to record data for a period of minutes—at least as long as you expect your measurement to be—to observe the decay of fluorescence because of any photochemistry of the target compound. It is also a good idea to change the excitation wavelength to the shortest you plan to use, and repeat this experiment to see how quickly it decays with short-wavelength excitation. If your target compound does exhibit oxygen sensitivity, then this photochemical study should be repeated with a purged sample. If photochemical decay is too rapid, the measurement protocol may need to be modified to limit the exposure of the sample to light during analysis. This can be done by blocking the excitation beam when not making measurements, and also by using shorter dwell times and taking larger wavelength steps. If available and practical, a flow cell can be used to decrease the amount of light exposure of the sample by constantly replacing it.

To test for a concentration dependence in your sample, dilute the target compound by a factor of 2, and compare the fluorescence excitation spectra of the original and diluted samples when scaled together. Their scaled profiles should be the same and not show the appearance or disappearance of peaks, while their raw intensities should differ by the dilution factor. If you are unsure whether the profiles are sufficiently alike, dilute again to see if there is a systematic change occurring in the spectra with concentration. If there is no systematic variation, the original concentration is fine to work with. If there is a systematic variation, then use a lower concentration that shows no further systematic variation.

If the sample concentration that is selected by the procedures of Part I and the preceding paragraph give a sample whose intensity exceeds the linear range of the instrument for the intended SBWs, the sample concentration can be reduced as necessary to remain in the linear range.

If your instrument has a temperature-regulated sample holder, then you can skip this step as long as the holder is working properly, and sufficient time is given for the sample to equilibrate. If your instrument has an unregulated sample, then prepare a cuvette with a septum covering it to protect it, and cool it in an ice bath. Set up the spectrofluorometer so it is ready for an immediate rapid measurement (short dwell times, larger steps in wavelength). Ensure the sample compartment of the spectrofluorometer is purged with dry gas to avoid condensation, and return the sample to the holder. Record a rapid fluorescence excitation spectrum and a rapid fluorescence emission spectrum, returning to the ice bath between measurements if needed. Then, block the excitation beam and give the sample a few minutes to warm to room temperature. When it has equilibrated, unblock the excitation beam and repeat the excitation and emission scans. These measurements will give you a semi-quantitative sense of how sensitive the sample is to temperature changes. When the actual sample measurements take place, ensure the cuvette has equilibrated and record the temperature of the sample or sample compartment during the measurement.

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Articles

Help for handling sample-specific concerns in spectrofluorometry.

Challenges of Spectrofluorometry Part 3: Sample-Specific Concerns

Part 1 of this three-part series provided a procedure for quickly recording acceptable fluorescence spectra with classic commercial spectrofluorometers for most samples in common 1-cm-pathlength cuvettes. Part 2 of the series describes instrument-specific concerns that require modifications to our initial procedure if we want spectra that can be accurately reproduced in other laboratories. Part 3 will discuss how the sample itself can make reproducibility between instruments challenging.

In Part I of this series, we described a simple procedure to select conditions under which fluorescence excitation and emission spectra are acquired with a commercial spectrofluorometer for the most common type of sample—a solution of emitting molecules in a common 1-cm-pathlength cuvette. This procedure is satisfactory for routine samples, particularly when the results are for relative or qualitative purposes. However, when comparisons are needed between different laboratories or different instruments, the differences between instrument responses become important to understand and rectify.

There are many reasons that spectrofluorometry of a routine sample might be inconsistent from instrument to instrument. This part of the series describes at a high level some of the reasons for these inconsistencies even when you “collect data right the first time,” and provides a procedure for how to correct for instrumental responses and artifacts.

Figure 1 in Part 1 provided a schematic diagram of a conventional spectrofluorometer with a continuous light source, and defines horizontal and vertical directions with respect to light polarization. For reproducible spectrofluorometry, we need to reproduce the instrument settings and filters at a minimum. However, even with these factors kept constant, there are reasons fluorescence data recorded on different instruments or at different times on the same instrument might be inconsistent with one another.

Beginning with the lamp and ending with the emission detector, every element of the instrument that produces, detects, or interacts with light can result in instrument-specific effects on a measured spectrum. Nearly all interactions can affect the light intensity or polarization to a greater or lesser degree.

Some of the instrumental factors that can affect the apparent spectrum and that were not discussed in Part I include the following:

The lamp intensity and spectrum can vary from instrument to instrument and over time

Transmission of light from the light source into the excitation monochromator can vary with wavelength and over time

The excitation spectrometer optics can affect the overall throughput and the polarization of light reaching the sample

The emission spectrometer optics will affect the throughput and polarization of fluorescence reaching the detector

The reference detector and its beam splitter have overall relative efficiencies that vary with wavelength and polarization of the beam

Any optical filters used in the system will introduce their spectral features, which may vary with the degree of collimation of the beam

Colored glass filters used in the spectrofluorometer can fluoresce and add to the background of the measurement (1)

Any interference-type filter used in the system will affect horizontal and vertical polarizations differently if not used at normal incidence

The cuvette affects reflection and transmission losses as a function of its material

The point of intersection of the excitation and detection optical paths inside the sample may vary, affecting the relative amount of inner filter effects that are observed

The cuvette positioning affects the point of overlap of the excitation and detection volumes inside the sample

A poorly chosen or poorly cleaned cuvette may have a measurable auto-fluorescence of its own, or may have a measurable absorption spectrum that interferes with the spectroscopy of the sample it contains (for example, optical glass in the UV)

Some cuvettes may not be intended for precision spectroscopy because of heterogeneities in the material

Using different sample geometries (90-degree, 180-degree, square or triangular cuvettes) will greatly affect self-absorption, polarization, and the sampling volume

The detectors have wavelength-dependent light detection efficiencies

Both monochromators may exhibit wavelength offset errors

Motor drive mechanisms could be malfunctioning leading to poor repeatability of the wavelength

Each monochromator may exhibit errors in spectral bandwidth (SBW)

The monochromator slits may not move evenly, causing the wavelength offset errors to vary with slit width

Gear mechanisms for moving gratings in both monochromators may exhibit hysteresis

Recorded spectra may have a background or intensity offset that varies with wavelength.

Reference detectors are commonly used to reduce the dependence of the spectrum on many of these factors in the excitation system. Pre-installed calibration files for the main detector may be used to largely correct for a number of these factors in the emission system. However, these compensations are not found universally, and even with them, there are factors that are not fully compensated or that change over time and require recalibration. As a result, we still observe wavelength- and polarization- dependent factors that vary between different instruments, or even between measurements made on the same instrument if parts have been replaced, if the alignment changes, if calibration files have been updated, removed, added, lost, or if components of the instrument have aged. In addition, slow phosphorescence of the sample could make a spectral profile dependent on how quickly the overall measurement is made, which could also vary between users or instruments.

Procedure: Evaluate → Collect → Correct

In Part 1, we described the method for choosing initial conditions for measurement. In Part 2, we focus on the process of modifying our initial conditions if necessary and correcting measurements for instrument response and artifacts. The procedure we are about to introduce is appropriate for situations in which you are trying to generate data for routine samples that will be reproducible on other instruments and in the future. We recommend that certain aspects (wavelength errors, SBW errors, range of linearity, polarization) of the instrument performance be determined before data is collected. By doing so ahead of data collection, final throughput correction procedures based on fluorescence standards and calibrated detectors can be applied properly.

We need to evaluate our system for wavelength errors in the excitation and emission monochromators, SBW errors, and detector linearity as a starting point to collecting data for interlaboratory comparisons. We also need to evaluate the need for polarization compensation in our instrument. These factors cannot be readily repaired after our corrected spectrum is created in the end.

Evaluation of the wavelength precision, SBW precision, and detector linearity are adapted from the literature (2), which provides much more information and is recommended to the reader.

A gear mechanism moves optical components of most monochromators. To avoid the effects of gear backlash or hysteresis on wavelength precision, all measurements should be recorded in the same scan direction (either increasing wavelength or decreasing wavelength). To ensure the wavelength of the first points in the spectrum are correct, we recommend over-scanning by a few nanometers. For example, if we are going to make our measurements by scanning downward in wavelength starting from 600 nm, we might want to begin the scan a few nanometers higher in wavelength so that gear backlash is removed by the time the monochromator reaches 600 nm. If scanning upward, we might start the scan a few nm lower in wavelength than the planned starting point for the same reason. Commercial instruments may automatically overscan the wavelength axis to avoid this problem.

Small atomic (mercury, neon, argon, krypton, xenon) lamps that are approximately the size and shape of pencils and that provide well-known reference wavelengths are common tools in spectroscopy. There are various manufacturers of these inexpensive lamps with different brand names. If a lamp of this type is placed in the position of the sample so that the emission monochromator entrance is illuminated, and if the entrance slit is narrowed to give the highest resolution, the apparent emission wavelengths of the atomic lines from these lamps can be measured and used to correct the emission monochromator wavelength axis. Any filters placed in the spectrofluorometer need to be removed before beginning.

These lamps are difficult to use to correct the excitation wavelength. One common approach to wavelength correction of the excitation monochromator is to use the emission monochromator as a reference. It requires the emission monochromator to have its wavelength axis determined first, and is only as good as the emission monochromator wavelength correction.

The first step in this procedure is to select an emission wavelength, place a cuvette containing an aqueous dilute scattering solution of your choice into the sample compartment, and record an excitation spectrum. Large peaks in the excitation spectrum will appear at the selected wavelength of the emission monochromator. Large peaks can also appear at 1⁄2 of that wavelength because of higher orders of the emission monochromator grating if those wavelengths are produced by a lamp. A few well-chosen emission monochromator settings can potentially provide several points for correction of the excitation monochromator wavelength axis. The accuracy of the emission wavelength correction is ±0.1 nm or better (2). The excitation wavelength correction done this way is not quite as good (±0.2 nm). Because of gear backlash, wavelength measurements should be recorded while scanning in the same direction that will be used in spectrofluorometry of the target compound.

The motors that drive the monochromators and slits are typically stepper motors, which require a driver to sequence the phases of the motor. If a phase of the motor fails, or if the driver board becomes intermittent, or if a connection becomes intermittent, it is possible for the driver to step inaccurately. A symptom of this failing would be a change in the sound of the instrument as it changes wavelengths, or an inability to return to the same wavelength. Some instruments may combine motors with an indexer that reads the position of the motors independently; if so, then the indexer may be susceptible to mechanical or electrical failures, also leading to poor repeatability. Repeatability of the monochromators can be tested by repeating the calibration runs several times and overlaying the scans to look for “jitter” in the peak positions. Poor repeatability is cause for an immediate call to the manufacturer.

The slit width is the main variable used to determine the SBW of a monochromator. The slits are formed as a space between two metal plates that each resemble knife-edges; the slits are each supposed to travel the same distance from the closed position so that the center wavelength is always the same regardless of SBW. The same pencil-type lamp used above can be used to determine the SBW of the emission monochromator by assuming the observed width of isolated atomic lines are solely because of the SBW of the monochromator. In this case, SBW is determined as full-width at half maximum (fwhm) of a measured peak. The excitation monochromator again requires a different procedure. By setting the SBW of the emission monochromator to its lowest setting, the SBW of the excitation monochromator can be estimated by recording an excitation spectrum of a dilute scattering solution and repeating the fwhm procedure. These procedures are thought to be subject to an uncertainty of ±0.5 nm in SBW (2). This test should be performed at a few different wavelengths to understand how any SBW error varies with wavelength. If there is any significant error in SBW, and particularly if the SBW error is a function of SBW, one possibility is that at least one side of the mechanical slit is not moving properly. If no fix or repair is possible, the wavelength error determined may be found to be a function of SBW; it is also likely the SBW will not be repeatable if the slit is not moving properly. As with wavelength unrepeatability, any indication of a SBW unrepeatability is cause for an immediate call to the manufacturer.

Even the best detectors become nonlinear at sufficiently high light intensities. Therefore, it is important to know the linear range over which your instrument operates. One approach is to measure a series of known concentrations of a fluorophore whose emission wavelength is similar to your target compound. To avoid nonlinearity from inner filter effects, the concentration of these standards should be kept very low (below 0.05 absorbance units). The peak excitation and emission wavelengths of the standards can be used, and the series of measured intensities can be plotted and fit to ensure they are consistent with the blank and show no systematic deviation from a straight line. If the highest detected signal is still linear, the calibration measurements can be repeated with an excitation SBW up to the limit described in Part I of this series, and an even wider SBW for the emission monochromator, to give a wide intensity range for the standard concentrations. The linearity of a detector, such as a photomultiplier tube, may change for different accelerating voltages, so care should be taken to use the detector as intended for your study. When changing the contents of a cuvette, the cuvette should be emptied, rinsed with clean solvent, rinsed with the next sample, emptied again, and then refilled before the next measurement. This procedure avoids cross-contamination and dilution of the sample.

If the fluorescence of a sample in the 90-degree sample geometry is at least partially polarized, there will be effects on its measured intensity and probably at least its measured excitation spectrum. Counterintuitively, this is true even if there are no polarization artifacts in any of the optics. Fortunately, polarized fluorescence is not generally a problem for small molecules in low viscosity solvents. However, even small molecules can exhibit polarized fluorescence if their fluorescence lifetime is short enough (3), whereas large molecules and viscous solvents invite this problem. If you are concerned that you might need to eliminate fluorescence polarization as a complication, then you can add a “vertical” polarizer in the same location as the excitation filter, and insert another polarizer in the same location as the emission filter to serve as a polarization analyzer. This orientation of the emission polarizer should be set to the 54.7° magic angle to make the measurement independent of the fluorescence polarization of the sample (4).

The conditions for measurement can be determined using the methods of Part I, or they can be optimized from that starting point. The following points are specific to measurements made for publication and reproducibility:

The conditions of measurement must be controlled and recorded. This includes making sure the instrument scan range, step size, spectral bandwidth, number of points per unit wavelength, and integration or dwell time, are appropriate for the sample and that they are all recorded and preserved. It also means that any steps taken to evaluate the wavelength accuracy, spectral bandwidth accuracy, photometric accuracy, and to prevent dependence on sample polarization, should be recorded and preserved

The clean sample cuvette should be handled with gloves and carefully wiped on all its optical surfaces with a fresh lint-free paper or cloth to remove dust or surface contaminants before being placed into the spectrofluorometer for measurements

If your instrument has an automated shutter to block the excitation beam, this should be engaged any time a measurement is not being made

A background spectrum of the pure solvent should be recorded under the same conditions as the sample. Ensure that the same cuvette is used for the background and for the sample, and that it is placed into the instrument in the same orientation. This procedure helps to account for any stray light, dark signal, amplifier switching, or filter fluorescence that might take place inside the spectrofluorometer, and can be subtracted from the measured spectrum before corrections are applied.

At this point, all concerns about critical aspects of the instrument that cannot be easily corrected post-measurement have been addressed. What remains is the part of the instrument performance that affects the throughput of the excitation source and the emission of their respective monochromators. On the excitation side, the reference detector, if present, corrects for many of the wavelength-dependent behaviors of the instrumentation. Without the reference detector, fluorescence excitation spectra could be so heavily convolved with the instrument response they would be almost uninterpretable without correction. However, the response of the reference detector itself and the effects of optical components after the reference detector still require throughput corrections.

There are several alternate procedures for implementing these throughput corrections and we describe only the simplest. At present, excitation and emission spectra require different approaches for their correction. The National Institutes of Standards and Technology (NIST) and the Federal Institute for Materials Research and Testing in Germany (BAM) are both excellent sources for details on instrument concerns and corrections that we recommend to the reader (1,4).

For most users, certified reference materials or secondary emission standards are the simplest approaches to correcting emission spectra. An excellent alternative is to use a calibrated lamp, but the standards offer the opportunity to use a cuvette-type sample.

A certified reference material is either a solution or glass that can be placed into a spectrofluorometer cuvette holder, and that has known excitation or emission properties. These materials are certified by, for instance, NIST in the United States and equivalent organizations in other countries. As an example, NIST has certified a series of standard reference materials that include solid glass, cuvette-shaped standards that can be placed directly into a spectrofluorometer (SRM 2943 and SRM 2944 for the spectral ranges 350–640 nm and 530–830 nm, respectively). Certified reference materials are relatively expensive compared to available secondary emission standards (5), though both are used in the same way for the same purpose.

Whether a certified reference material or a secondary standard, what these reference materials have in common is a defined emission spectrum under defined measurement conditions. The measured emission spectrum of the reference material in your instrument is compared to its certified or reported spectrum to determine a correction factor over the range of wavelengths for which it has been evaluated. This correction factor is then used to correct the measured spectrum of your target fluorescence sample.

There are not currently any primary fluorescence excitation spectroscopy standards like those for fluorescence emission spectroscopy. The simplest available approach to correcting fluorescence excitation spectra is to use a calibrated detector. In this case, the detector is placed in the location of the cuvette to intercept the excitation beam, and its corrected signal is recorded as a function of excitation wavelength. The reference signal of the spectrofluorometer is recorded at the same time. The ratio of these two responses then becomes a correction factor for the wavelength range over which the detector has been calibrated. If there is no reference channel, the corrected signal of the calibrated detector is used to form the correction factor by itself.

Although this article covers spectrofluorometry only at a very high level without much detail, it is still restricted to only one measurement case (solution cuvettes) in steady-state spectrofluorometers. The wide field of spectrofluorometry includes many more sample types and configurations, along with a wide variety of instrumentation, all suited to particular purposes. If the authors could leave the reader with two important messages, the first would be this: Read the user manual for your instrument, read literature about your measurement type, and read about your sample type to prepare yourself for making your own measurements. Having this background knowledge ahead of time will enable you to avoid as many errors as possible and enable you to compare your own measurements to those in the literature to assess whether your measurements are reasonable or not. The second message is that there are many parameters that should be recorded and transmitted about spectrofluorometry measurements in addition to the basics of the instrument settings, without which reproducing your spectra could become very difficult for others.

(2) P.C. DeRose, “Standard Guide to Fluorescence – Instrument Calibration and Validation,” NIST Interagency/Internal Report (NISTIR) – 7458 (National Institute of Standards and Technology, Gaithersburg, MD, 2008, accessed September 27, 2021). https://doi.org/10.6028/NIST.IR.7458

Standardization and Quality Assurance in Fluorescence Measurements I

 (Springer-Verlag, Berlin, 2008), vol. 5.

, 1179–1189 (1998). https://doi.org/10.1366/0003702981945192.

In the second part of a three-part series, instrument-specific concerns that require modifications to reproduce fluorescence spectra accurately are addressed.

Challenges of Spectrofluorometry, Part 2: Instrument-Specific Concerns

This article is the first of a 3-part series that gives a procedure to avoid common errors in spectrofluorometry using an example case. Part 2 will focus on throughput and polarization correction of spectrofluorometers for the purpose of inter-laboratory comparisons, while Part 3 will focus on non-ideal sample behavior.

In this first of a three-part series, we address the challenge of collecting good quality data by recommending procedures designed to “collect data right the first time." We focus our attention on the two most common fluorescence spectroscopies. The first is fluorescence emission spectroscopy, in which a single excitation wavelength is used and fluorescence is measured as a function of emission wavelength. The second is fluorescence excitation spectroscopy, in which a single emission wavelength is used to measure how well fluorescence is excited as a function of excitation wavelength. We assume the case in which the user plans to record both types of spectra for a compound in a solution.

All spectrofluorometers have a light source with a wavelength selector, optics that transfer the light to a sample, optics that pick up light from the sample, a wavelength selector for detection, and one or multiple light detectors. A common instrument layout for the optical bench of a spectrofluorometer is shown in Figure 1.

FIGURE 1: Instrument layout for a common commercial spectrofluorometer. A xenon arc lamp is illustrated as the light source that is focused into an excitation monochromator. This unit selects a narrow excitation wavelength band that is focused into a sample in a cuvette. Fluorescence is emitted at longer wavelengths and directed into an emission monochromator, where a narrow emission wavelength band reaches a detector (Det). A reference photomultiplier tube (Ref) measures a fraction of the excitation light to correct for excitation source intensity, and a bandpass filter (BPF) and longpass filter (LPF) prevent stray light from the excitation monochromator from being detected. The 90-degree angle between rays entering the sample and the fluorescence emerging toward the detector defines a horizontal plane.All light rays polarized in this plane at the sample are called horizontally polarized, while rays polarized perpendicularly to this plane at the sample are called vertically polarized.

In a classic instrument, the light source might be a broadband high-pressure xenon arc lamp. For this type of light source, a housing with mirror optics and a condenser lens are used to transfer the brightest part of the emission from the arc to the entrance slit of an excitation monochromator. The excitation monochromator selects mostly a narrow band of wavelengths that exit toward the sample. Many instruments include a pick-off mirror in this region that directs light to a reference detector (often a photomultiplier tube) to compensate for the varying intensity of light at each wavelength emerging from the excitation monochromator. Most spectrofluorometers have a provision for a bandpass filter (BPF) to clean up the excitation light, after which the excitation then encounters a sample. The sample is often a solution of an analyte in a 1-cm pathlength fluorescence cuvette.

Fluorescence from the sample emerging at right angles to the excitation is collected by more optics. A longpass filter (LPF) is frequently used here to remove any scattered excitation light from the emission before it focuses on the entrance slit of an emission monochromator. Wavelengths are again selected and a narrow band of emission wavelengths is focused to a detector, which is often a red-enhanced photomultiplier tube.

The most common substantial errors we observe in spectrofluorometry are as follows: a) poor choices of excitation and emission wavelengths leading to noisy spectra; b) a poor choice of the sample concentration leading to inner-filter effects; c) a poor choice of the spectral bandwidth (SBW) and step size leading to an unfaithful reproduction of the true spectrum, or long, noisy measurements; d) a poor choice of sample cell or solvent that obstructs the analyte spectrum; and e) detecting scattered light that leads to artifacts in the spectra.

Procedure: Absorption → Emission → Excitation

You can avoid all of these problems by beginning studies with a good quality absorption spectrum of the analyte recorded on a spectrophotometer using the same sample cuvette you intend to use for spectrofluorometry, ideally with a SBW of 2 nm or better. This measurement helps you select the best choices for excitation wavelengths, the likely range for emission wavelengths, an appropriate concentration, and an appropriate SBW and step size. It confirms that the sample cell and solvent are suitable. Finally, it allows you to select conditions that avoid most problems with scattered light.

If you are planning to record fluorescence emission and excitation spectra for a compound in solution, the best procedure for getting your data right the first time is to do the following in order:

use a spectrophotometer to collect an absorption spectrum of the sample

record a fluorescence emission spectrum of the sample based on information in the absorption spectrum

record a fluorescence excitation spectrum based on the information in the absorption and fluorescence emission spectra.   

As an example, let us consider the following absorption and fluorescence spectra (Figure 2) for tetrakis(4-carboxyphenyl)porphyrin (TCPP). Consider the absorption spectrum recorded in our intended fluorescence cuvette and solvent first. The fact that we can see the absorption spectrum tells us that ethanol and the quartz sample cell are appropriate for the measurement through this range of wavelengths. If the solvent or cuvette absorb light, the absorption spectrum will either go off-scale or will be nothing but random noise.

FIGURE 2: (Blue solid line) Absorption and (red solid line) fluorescence emission spectra of tetrakis(4-carboxyphenyl)porphyrin in ethanol in a quartz fluorescence cuvette. The absorption spectrum multiplied by 20 is shown as a dashed blue line.

The absorption spectrum itself tells us that we could potentially excite TCPP at most wavelengths below about 660 nm because it has absorption throughout the region, although the best initial choice of excitation wavelength is likely near 417 nm because that is where the strongest absorption is.

Our next step would be to record a fluorescence emission spectrum using our selected excitation wavelength. The absorption spectrum does not give us direct information on the wavelength range where the strongest emission will occur in our fluorescence or phosphorescence spectrum. But even this is guided by our absorption spectrum, because it tells us what wavelength range to consider.

The longest wavelength absorption we see for TCPP has a maximum near 645 nm; our fluorescence will likely not be at a significantly shorter wavelength (in fact this molecule has its apparent emission maximum at nearly the same wavelength, which is unusual). Because our selected excitation wavelength of 417 nm is pretty far below 645 nm, we might suggest recording our first fluorescence emission spectrum beginning at 600 nm to start.

The absorption spectrum also tells us what concentration of analyte to use. The peak absorbance at 417 nm in Figure 2 is near 

 = 0.42. That is far too high, and as a result, we should dilute the sample so that its peak absorbance is in the range 

 = 0.05–0.1 before spectrofluorometry, which avoids a phenomenon known as an 

The absorption spectrum further helps us choose the SBW for our fluorescence emission spectrum. In Figure 2, we observe that the sharpest spectral feature has a full width at half maximum (fwhm) near 10 nm. This is much broader than the SBW of the spectrophotometer we used for the absorption measurement, so we can feel confident this represents something inherent to our analyte. We should not use any SBW broader than that, and probably should start a little better—for instance, a SBW of 5 nm. As a first guess, we can use a SBW of 5 nm for both the emission monochromator and the excitation monochromator. The maximum step size for the measurement follows directly from our SBW: We should record a minimum of two points in each SBW. If the SBW is 5 nm, it is convenient to choose a step size of 1 or 2 nm.

The absorption spectrum also helps us address the issue of scattered light. There are two types of scattered light problems that we face, but the first and most obvious is because of the second-order transmission of light in the emission monochromator. For example, if your emission monochromator is set at a wavelength of 834 nm, this means it transmits light at a wavelength of 834 nm to the detector in the first order; however, it can also transmit light at 417 nm in the second order. Running our fluorescence emission spectrum through 834 nm could produce a giant spike in our spectrum because of scattered excitation that could easily saturate our detector. The second, less obvious scattered light problem is stray light in our excitation and emission monochromators. This stray light can cause a baseline artifact, which could be important if the spectrum is weak.

Both of these stray light problems can be avoided with optical filters that clean up out-of-band light from your excitation source and prevent the excitation from reaching your emission monochromator. A BPF that transmits 417 nm light, but blocks shorter and longer wavelengths, and a LPF that blocks 417 nm light, but transmits 600 nm wavelength light and beyond, will prevent scattered light from impacting the measurement at all. If you only have a longpass filter, that is still helpful; if you have no filters at all—and many spectrofluorometers do not come with them automatically—then avoid running the emission monochromator to a wavelength that is an integer multiple of the excitation wavelength.

We’re now ready to record our fluorescence emission spectrum, shown in Figure 2 as a red curve.

Our fluorescence emission and absorption spectra can then help us decide how to record a fluorescence excitation spectrum. For example, in our fluorescence emission spectrum of TCPP above, there are two large fluorescence maxima, one at 646 nm and another at 713 nm. But the absorption spectrum tells us that our fluorescence excitation spectrum will likely continue to ~660 nm. It makes sense then to choose 713 nm as our detection wavelength and record our excitation spectrum from 360 nm (below that could potentially produce a second-order peak) to 670 nm. A LPF that transmits 713 nm but blocks below 670 nm will help us avoid most light scattering problems, and we have already selected a SBW and step size for our measurement, so a good quality fluorescence excitation spectrum can then be acquired.

If you decide you want better data than this procedure provides, you can return and adjust the SBW separately for excitation and emission spectra. For example, the SBW for the excitation source can usually be increased when you are recording a fluorescence emission spectrum, and the SBW for the emission detector can be increased when you are recording a fluorescence excitation spectrum. In addition, the integration time or dwell time at each measurement point can be increased from the default offered in commercial software. Each of these changes will increase the measurement signal-to-noise ratio.

The procedure we provided here will allow you to record good quality spectra in most cases in your first measurement. Part 2 of this three-part series will focus on generating data that can be reproduced on different instruments. Part 3 will discuss how some samples can be responsible for making data irreproducible, even when everything else was "done right the first time".

 (Springer, Boston, Massachusetts, 3rd Ed., 2006).

 (Prentice-Hall Inc., Englewood Cliffs, New Jersey, 1988).

Analysts using fluorescence emission and fluorescence excitation spectroscopy may encounter several common problems in their measurements. This tutorial, the first of a three-part series, provides a procedure to help avoid them.

Zechariah Kitzhaber

Articles

Challenges of Spectrofluorometry Part 3: Sample-Specific Concerns

Challenges of Spectrofluorometry, Part 2: Instrument-Specific Concerns

Challenges of Spectrofluorometry, Part 1: Collect Data Right the First Time