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á1853ñ FLUORESCENCE SPECTROSCOPY—THEORY AND PRACTICE

THEORY

Fluorescence is a two-step process that requires an initial absorption of light followed by emission. [NOTE—Many terms and
variables used in this general chapter are explained in the Glossary.] Fluorescence spectroscopy is an electronic spectroscopic
method related to ultraviolet–visible–near infrared (UV–Vis–NIR) absorption spectroscopy. It is also a background-free method
that involves light emitted from the sample in all directions, as is the case with Raman spectroscopy. The initial absorption of a
photon by a molecule in the sample promotes an electron to an excited state. The excited electron returns to the ground
electronic state by emitting a photon. If the emission arises from an “allowed” transition that typically has a short lifetime
between 1 ns and 10 ns, then it is called fluorescence. If the emission arises from a “forbidden” transition that typically has a
long lifetime between 1 ms and 1 s, then it is called phosphorescence. Under similar conditions phosphorescence usually is less
intense than fluorescence. This general chapter discusses fluorescence spectroscopy, but many points raised here also apply to
phosphorescence. The basic concepts behind fluorescence spectroscopy have been well established, but its applications and
standardization are still expanding and progressing, making it a developing rather than a mature method.
The most common type of fluorescent sample is a dilute, transparent solution that absorbs light following the Beer–Lambert
Law and that emits a corresponding fluorescence intensity that is directly proportional to the concentration, the absorptivity,
and the fluorescence quantum yield of the fluorescent species or fluorophore. A conventional fluorescence spectrometer has
both excitation and emission wavelength selectors. It collects a spectrum by fixing the wavelength of one of the selectors and
scanning the other wavelength selector over a range. When the excitation wavelength is fixed and the emission wavelength is

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scanned, the resulting spectrum is termed an emission spectrum. When the emission wavelength is fixed and the excitation
wavelength is scanned, the resulting spectrum is termed an excitation spectrum (Figure 1). The fluorescence spectrum is plotted
as relative intensity or counted photons of fluorescence vs. wavelength. The appearance of a fluorescence spectrum is much
like a UV–Vis–NIR absorption spectrum. In fact, the shape or contour of an excitation spectrum often is identical to that of the
corresponding absorption spectrum for an organic dye in solution over the same wavelength range.
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Figure 1. Fluorescence excitation and emission spectra for fluorescein in borate buffer. The wavelength axis shows excitation
and emission wavelengths.

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Polyatomic fluorophores in condensed media (e.g., solutions, thin films, and solids at room temperature) exist in ground or
excited electronic states in a broad distribution of vibrational energy levels and cause homogeneous broadening of excitation
or emission spectra, respectively. A microenvironment or shell also surrounds each fluorophore in condensed media, and
differences in the structures of these shells among individual fluorophores cause inhomogeneous broadening. These two types
of broadening cause fluorescence spectra to be broader than some other types of spectra (e.g., mid-infrared or Raman spectra).
The typical width of a fluorescence band is between 10 nm and 100 nm. Once it is electronically excited, a polyatomic
fluorophore experiences vibrational relaxation before emitting a photon, causing a red shift or Stokes shift of the fluorescence
spectrum relative to the wavelength at which it was excited.
Few naturally occurring biological compounds fluoresce strongly. However, the development of a large array of fluorescent
indicator dyes, used mainly to bind to ions or indicate pH or polarity, has led to increased interest in the use of both direct and
secondary fluorescence techniques, e.g., fluorescence resonance energy transfer (FRET). While many of these probes inherently
do not have very high quantum yields, their fluorescence changes greatly upon binding and upon the associated solution
chemistry. For instance, the pH of the sample solution is an important factor to control when considering not only its impact
on the fluorescence of the probe, but also on the detection of other possible fluorescing of fluorescent dye binding species.
Some fluorescent probes, such as fluorescein (pH dependent) and rhodamine and its derivatives, have very large absorptivities
and quantum yields approaching one—i.e., they fluoresce nearly as many photons as they absorb. Fluorescence methods are
also termed background-free because very little excitation light reaches the detector. These advantages make fluorescence
detection highly sensitive, down to single-molecule detection in some cases. Specificity and sensitivity are two of the more
important strengths of fluorescence methods. Fluorescence spectroscopy also typically is not destructive to the sample, and
measurements can be made quickly (on the order of seconds to minutes).
A right-angle or 0°/90° geometry often is used to measure dilute solutions and other transparent samples. In such cases, the
excitation beam is normal to the sample, and fluorescence is detected at a 90° angle relative to the beam (Figure 2a). A front-face
geometry is used to measure optically dense samples where the excitation beam is incident on the sample at <90° and the

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fluorescence is collected at an angle ≤90° (Figure 2b). The epifluorescence geometry is a special case of the front-face geometry
that often is used in fluorescence microscopy and optical fiber–based fluorometers. In epifluorescence geometry the excitation
beam and collected fluorescence are both normal to and are on the same side of the sample, i.e., a 0°/0° geometry. A 0°/180°
transmitting geometry often is used in microscopy (Figure 2c).ci
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Figure 2. Schematic of the excitation beam (EX) and detected emission (EM) orientations for (a) 0°/90° right-angle
transmitting, (b) front-face, and (c) 0°/180° transmitting geometries.

The number of chemical assays and screening methods using fluorescence detection continues to increase rapidly and has
resulted in a corresponding increase in the need for standardization of fluorescence measurements. Only a few standard
methods and reference materials have been well established and are readily available at present for the characterization of
fluorescence measuring systems. National metrology institutes and international standards organizations are working to provide
new fluorescence standard materials and methods in the near future. This general chapter briefly discusses the major issues that
should be considered by users of fluorescence instruments who aim to achieve high-quality measurements. Standard methods
and materials are also described where appropriate. A few guidelines and recommendations have appeared, but this general
chapter aims to be most useful to nonexpert users of fluorescence spectrometers.

INSTRUMENTATION

All modern fluorescence measurements involve irradiating the sample with the beam from a suitable light source, selecting
the excitation wavelength, collecting the resulting fluorescence, rejecting the Rayleigh-scattered light, selecting the emission

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wavelength, and detecting the fluorescence signal. The following functions will be discussed individually, along with the
equipment used to achieve these functions in commercial instruments:
1. excitation light source
2. excitation wavelength selector
3. sampling device
4. emission wavelength selector
5. detector.

Excitation Light Source

A variety of lamps, lasers, and light-emitting diodes (LEDs) are used as excitation sources. Continuous and pulsed versions
of these sources are used for steady-state and time-resolved instruments, respectively. Xenon lamps are the most commonly
used because of their relatively high intensity and broad wavelength range (UV to NIR). Lasers are the highest-intensity sources
and are used in applications where short collection times and small amounts of sample are required, e.g., for flow cytometry
or microarrays.

Excitation Wavelength Selector

The intensity of scattered light at the excitation wavelength (i.e., Rayleigh scattering) can be comparable to or greater than
that of the fluorescence at the sample. Therefore, the excitation wavelength profile should not overlap the emission wavelength
region being detected. This is achieved for lamps by using an excitation wavelength selector (e.g., a filter or a monochromator

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with a known peak transmission wavelength and bandwidth) between the lamp and sample. The inherent bandwidth of the
radiation from a laser or an LED often is narrow enough that an excitation wavelength selector is not necessary. This selector
also enables fluorescence excitation spectra to be resolved.

Sampling Device
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The sampling device includes all optics and other equipment needed to deliver the excitation beam to the sample, collect
the emission from the sample, and hold the sample in place. Sample formats include cuvettes, microwell plates, microarrays,
microscope slides, and flow systems and may be accompanied by a variety of optical delivery and collection systems, including
conventional transmitting, front-face, and epifluorescence systems and fiber optic–based probes.
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Emission Wavelength Selector
As with the selector for excitation, the emission wavelength selector helps to ensure that the emission wavelength region
being detected does not overlap with the excitation wavelength profile. This approach enables individual fluorescence bands
to be detected when multiple bands are present and allows fluorescence emission spectra to be resolved. Emission wavelength
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selectors also are important for the rejection of stray light. Filters, monochromators, and grating polychromators often are used
for emission wavelength selection.

Detector
For the detection of emitted light, a photomultiplier tube (PMT) or a charge-coupled device (CCD) array is placed after the
emission wavelength selector. The detection of the excitation beam in order to monitor its intensity commonly is done by a
quantum counter detector or a photodiode placed before the sample and to which a small fraction of the excitation beam is
split off from the rest.

FACTORS THAT AFFECT QUANTITATION

Instrument-Based Factors
Measurements on a fluorescence instrument require that instrument parameters such as wavelengths, bandwidths, and
detector gain be set. All of these parameters can be set with varying degrees of repeatability and accuracy, depending on the
instrument used. These factors can introduce measurement uncertainty or bias that is particularly significant when measured
values are compared between instruments. For instance, the measured peak positions of the emission bands of two analytes
may differ between instruments because of a wavelength bias. A corresponding bias between instruments could be introduced
in the results of an assay that depends on the ratio of the fluorescence intensities at the two specified emission wavelengths.
The intensity of the excitation beam can change significantly with excitation wavelength or with time because of the
wavelength dependence of the intensity of the light source and the transmittance of the excitation wavelength selector or the
time dependence of the light source intensity. Thus analysts should monitor the excitation beam intensity and correct the
measured fluorescence intensity for these fluctuations. This monitoring can be particularly important when excitation spectra
are collected because the excitation intensity often has sharp peaks and valleys when lamp sources such as a xenon (Xe) lamp
are used.
The responsivity of a detection system is not linear with intensity at all intensities, so analysts should know the linear intensity
range of the detection system used. The linear range for most detection systems ranges from its limit of detection up to a

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threshold intensity above which the responsivity becomes increasingly nonlinear with increasing intensity. Analysts should
establish the linear range of the fluorescence detection system before they attempt to calibrate the responsivity of the detection
system.
The responsivity of the detection system also is a result of the wavelength dependence of the transmittance of the emission
wavelength selector and the responsivity of the detector. These factors can affect the shape of a measured emission spectrum.
The diffraction efficiency of gratings and the responsivity of detectors often depend on polarization. Changes to instrumental
polarization settings can result in changes in the observed excitation intensity and the responsivity of the detection system.
Even when polarizers are not used within the instrument, the excitation beam may be polarized by the optical system itself and
may affect the responsivity of the detection system, and is instrument dependent. In addition, emission polarization effects not
only can cause intensity differences but also can change spectral correction factors.
The passing of multiple wavelengths by a diffraction grating can introduce unexpected sharp peaks into a fluorescence
spectrum. So that incident light is diffracted at a desired wavelength, a grating equation is used to set the angle of the grating
with respect to incident light:

mλ = d(sin α + sin β), m = 0, 1, 2, ...

m = diffraction order
d = groove spacing on the grating
α = angle of the incident wavefront relative to the grating normal
β = angle of the diffracted wavefront relative to the grating normal
The value of mλ, not λ, is fixed, where m is an integer termed the diffraction order. Therefore, the grating equation can be
satisfied by more than one wavelength for a single grating position. For instance, if a grating in an emission monochromator

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is set to pass 500-nm light at first order, it also will pass 250-nm light at second order. As a result, the scattered light from a
250-nm excitation beam will be detected as a peak at an emission wavelength of 500 nm unless a suitable optical filter is inserted
in the beam.

Sample-Based Factors
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The fluorescence intensity of optically dense samples (e.g., absorbance A >0.05 at a path length of 1 cm) does not increase
linearly with concentration because of significant absorption of the excitation beam and/or emission (reabsorption) by the
sample. These inner filter effects also can greatly reduce the amount of fluorescence that reaches the detector, especially when a
right-angle transmitting geometry is used. The fluorescence intensity can become strongly dependent on sample position and
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optical geometry. At even higher concentrations, aggregation of fluorophores often occurs, causing the shape of the
fluorescence spectrum to be different from that of a dilute sample and also causing nonlinear concentration behavior.
The fluorescence intensity of a sample may decrease with time of exposure to light because of photobleaching and
photodegradation. This is particularly true of most organic dyes, which are the most widely used fluorescent probes. Analysts
should limit the time that such samples are exposed to light in order to obtain reproducible fluorescence intensities and in some
cases even reproducible spectral shapes.
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The fluorescence intensity of fluorophores is temperature dependent. Typically, the rates of fluorescence quenching
processes, such as collisional quenching in solutions, increase with temperature and cause a decrease in fluorescence intensity.
Temperature coefficients for fluorescence intensity for particular fluorophores can be used to correct for this temperature
dependence.
The absorbance and consequently the intensity of fluorescence from a sample depend on the orientation of the sample’s
absorption transition dipole with respect to the polarization of the excitation light. The polarization of fluorescence is parallel
to the direction of polarization of the fluorescent species’ emission transition dipole. Fluorescence polarization is parallel to the
orientation of the fluorescent species’ emission transition dipole. Fluorescence anisotropy (r) is used to describe the extent of
polarization of emission and is defined by:

r = (I|| − I⊥)/(I|| + 2I⊥)

I|| = observed fluorescence intensity when the fluorometer’s emission polarizer is oriented parallel to the direction of the
polarized excitation
I⊥ = observed fluorescence intensity when the fluorometer’s emission polarizer is oriented perpendicular to the direction
of the polarized excitation
A sample whose fluorophores are oriented nonrandomly and have a rotational period that is long compared to their
fluorescence lifetime will emit anisotropic fluorescence. The spectral shape and intensity of such fluorescence depend on the
viewing angle and the instrument’s polarization factors.
A fluorophore’s fluorescence intensity and peak position, and sometimes even its spectral shape, often depend on the
environment, including changes caused by the solvent used, the solution’s pH, or the species to which the fluorophore is bound.
For the above reasons care should be taken during the experimental design of a new methodology to consider and evaluate
these effects, and matrix match the reference and unknown as appropriate.
A Raman signal can introduce peaks into the fluorescence spectrum. The Raman peaks of the sample’s solvent or matrix are
those most commonly encountered. For instance, the Raman peak of water, which is found red-shifted 3382 cm−1 from the
chosen excitation wavelength, typically is observed in the fluorescence spectrum of any aqueous solution excited by UV or blue
light.

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CALIBRATION OF FLUORESCENCE INSTRUMENTS

Two types of fluorescence instrument calibrations are used. The first and most commonly used is analyte specific and
determines the relationship between the response of the instrument (fluorescence intensity) and the concentration or amount
of a specific analyte. The second is analyte independent and is intended for spectral instruments. In this case, the wavelength
accuracy for emission and excitation and the spectral responsivity of the detection system are calibrated across the entire or a
continuous part of the wavelength range of the instrument.

Analyte Concentration—Calibration Curves

Calibration curves of instrument response (fluorescence intensity) vs. analyte concentration are determined using reference
materials that contain the analyte of interest. For instance, the fluorescence intensities of a set of solutions at different, known
analyte concentrations that cover a desirable concentration range can be measured and plotted vs. concentration. The plot
then is fitted to a polynomial, typically a straight line. The resulting calibration curve is both analyte and instrument specific
and can be used to determine analyte concentrations of unknown samples. This type of calibration may not be accurate when
the microenvironment surrounding the fluorophore is different in the reference and unknown samples. In addition, users must
ensure that the fluorescence intensities of samples are reproducible and do not decrease over the time when they are being
excited and measured because the organic dyes typically used may be prone to photobleaching.
In some cases, calibration samples at known concentrations and prepared from appropriate reference materials may not be
available. Firstly, organic dyes that are used as fluorescent probes often are not commercially available at a known high purity
that enables production of reference solutions. Secondly, in complex systems where fluorophores are bound to large molecules,

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cells, or microbeads, the concentration of bound fluorophores in a solution or suspension may be difficult to determine. In the
latter case, the molecules of equivalent soluble fluorophore (MESF) scale has been proposed as an alternative way to use
calibration curves to quantify fluorescence intensity for a particular analyte.

Emission Wavelength and Spectral Slit Width

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A variety of reference standards have been proposed for use in the determination of emission wavelength accuracy, including
atomic lamps and inorganic and organic fluorophores. The most widely used and best characterized of these are low-pressure
atomic lamps, commonly termed pen lamps. In this case, the type of pen lamp (e.g., Hg, Xe, etc.) is chosen so that its radiated
atomic lines are within the desired wavelength range. The lamp is placed at the sample position so that its light is centered in
the optical path of the detection system of the instrument. The accuracy of this method may decrease if the pen lamp is not
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properly aligned. The emission wavelength selector–detector then measures the signal over the wavelength range of interest.
The measured wavelength positions of the resulting sharp peaks then are compared with the known positions to determine
wavelength accuracy.
The spectral slit width accuracy of the emission wavelength selector can be confirmed by measuring the spectral bandwidth,
taken to be the full width at half the peak maximum, of a single line of a pen lamp. For fluorescence spectrometers with both
excitation and emission monochromators, an alternative method can be used when one monochromator is scanned over the
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position of the other.

Excitation Wavelength and Spectral Slit Width

Many of the reference samples that are used for determining emission wavelength accuracy also can be used for excitation
wavelength accuracy. For instance, a pen lamp can be placed at the excitation light source position so that the resulting
spectrum is detected after the excitation wavelength selector using the instrument’s reference detector. However, in this case, a
relatively weak signal may limit the number of useful atomic lines, and therefore alignment of the pen lamp is more critical in
this instance than for the emission wavelength accuracy determination.
Once the accuracy of the emission wavelength selector has been determined, use a diffuse scatterer, e.g., a scattering solution
or a diffuse reflector, at the sample to scatter a fraction of the excitation beam into the detection system to determine excitation
wavelength accuracy. One wavelength selector is set at a fixed wavelength and the other is tuned over the same wavelength
to obtain a spectrum. The wavelength bias between the two wavelength selectors is equal to the difference between the set
wavelength position and the observed peak position of the collected spectrum. This method can be used at any wavelength,
unlike many other methods that depend on a limited number of set excitation wavelengths determined by the reference material
chosen. Methods similar to those used for spectral slit width accuracy of the emission wavelength selector also can be used to
determine the spectral slit width accuracy of the excitation wavelength selector.

Linearity of the Detection System

Several approaches are available to determine the detection system’s linear intensity range. They can be separated into three
types, based on the tools used to vary the intensity of light that reaches the detector: (1) double aperture, (2) optical filters
and/or polarizers, and (3) fluorophore concentrations. The double-aperture method is the best established and probably is the
most accurate when done correctly, but it also is the most difficult to perform. A variety of methods using optical filters,
polarizers, or a combination of the two have been reported. These methods require high-quality, often costly, components and
some user expertise. The third method is the most popular and is easiest. It uses a set of solutions obtained by serial dilution
of a fluorescent stock solution that is similar to one used for obtaining calibration curves for analyte concentration, as described
earlier. In this case, analysts use solutions with low concentration (A <0.05 at 1-cm path length), but fluorophore adsorption
to cuvette walls may affect measurements at very low concentrations. Users must ensure that the fluorescence intensities of

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samples are reproducible and do not decrease over the time that they are being excited and measured because the organic
dyes typically used may be prone to photobleaching.

Signal Level (Relative Emission)

Calibration of the relative responsivity of the emission detection system with emission wavelength, also referred to as spectral
correction of emission, is necessary for successful quantification when intensity ratios at different emission wavelengths are
compared or when the true shape or peak maximum position of an emission spectrum must be known. Such a calibration is
required because the relative spectral responsivity of a detection system can change significantly over its wavelength range
(Figure 3). Analysts should know the degree of photometric precision required for successful quantitation. The linear range of
the detection system is determined before this calibration is performed, so that appropriate steps are taken (e.g., the use of
attenuators) to ensure that all intensities measured during this calibration are within the linear range. When one uses an emission
polarizer, the spectral correction for emission depends on the polarizer setting.
Two methods are preferred for calibrating photometric responsivity: one (Method A) uses light from a calibrated source (CS),
and the other (Method B) uses certified reference materials (CRMs). Both give results that are traceable to national metrology
institutes. A calibrated tungsten white light source is used most commonly for Method A and covers the wavelength range from
about 350 nm into the NIR. Standard reference materials from the U.S. National Institute of Standards and Technology and
CRMs from the German Federal Institute for Materials Research and Testing currently are available for use in Method B. Corrected
emission spectra of some commonly used dyes also have been reported in the literature. Method A is more difficult to implement
than Method B and requires periodic recertification of the CS. A third method, Method C, uses a calibrated detector and a
calibrated diffuse reflector. This method typically has larger uncertainties than Method A and Method B but is recommended in
UV and NIR wavelength regions that are not covered by the other two methods.

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Figure 3. Example of the relative spectral responsivity of an emission detection system (PMT-based grating monochromator)
for which a correction must be applied to a measured emission spectrum to obtain the true spectral shape (relative intensities).

METHOD A
In Method A, analysts direct CS light into the emission detection system by placing the CS at the sample position. If the CS
is too large to be placed at the sample position, analysts can place a calibrated diffuse reflector (CR) at the sample position to
reflect the light from the CS into the emission detection system. The emission wavelength selector is scanned over the emission
region of interest using the same instrument settings as used with the sample, and the signal channel output (S″) is collected.

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The known radiance of the CS incident on the detection system (L) can be used to calculate the relative correction factor (CCS)
so that:

CCS = L/S″

CCS = relative correction factor

L = radiance of the CS incident on the detection system
S″ = signal channel output
The corrected emission intensity is equal to the product of the signal output of the sample and CCS.

METHOD B
In Method B, analysts place the fluorescence standard at the sample position. Its spectrum is collected and is compared to
the certified spectrum according to the instructions given on the accompanying certificate, which yields spectral correction
factors for the instrument.

METHOD C
Method C involves two steps: Step 1 uses a calibrated detector (CD) at the sample position to measure the flux of the excitation
beam as a function of excitation wavelength. Step 2 uses a CR to reflect a known fraction of the flux of the excitation beam
into the detection system. This is done by placing the CD at the sample position at a 45° angle, assuming a 0°/90° instrument
geometry, and synchronously scanning both the excitation and emission wavelength selectors over the emission region of

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interest while collecting both the signal output and the reference output. This method allows analysts to calculate the relative
correction factor. This method has larger uncertainties than those for Method A or Method B and typically is more difficult to
implement.

Reference Signal Level (Relative Excitation)

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Calibration of the excitation intensity with excitation wavelength is necessary for successful quantitation when analysts
compare intensity ratios at different excitation wavelengths or when analysts must know the true shape or peak maximum
position of an excitation spectrum. Such a calibration is necessary because the relative spectral flux of an excitation beam at
the sample can change extensively over its wavelength range (see Figure 4). The neglect of excitation intensity correction factors
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can cause even greater errors than neglect of emission correction factors. Fortunately, many fluorescence instruments have a
built-in reference detection system to monitor the intensity of the excitation beam. This monitoring usually is done using a
photodiode, PMT, CCD, or a quantum counter detector to measure a fraction of the excitation beam that is split off from the
rest of the beam. The collected reference signal can be used to correct the fluorescence signal for fluctuations caused by changes
in the excitation beam’s intensity. Reference detectors often are not calibrated with excitation wavelength, which introduces
errors that can be particularly large over longer excitation wavelength ranges (e.g., >50 nm) or in a wavelength region where
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the excitation intensity changes rapidly with excitation wavelength (e.g., the UV range). When an excitation polarizer is used,
the spectral correction for excitation intensity depends on the polarizer setting.

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Figure 4. Example of the relative flux of an excitation beam (Xe lamp grating monochromator) for which a correction must
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be applied to a measured excitation spectrum in order to obtain its true spectral shape.

When a reference detector is not built into an instrument, a spectral correction for the reference channel or an independent
spectral correction of excitation intensity is required. Three methods can be used to determine the spectral correction of
excitation intensity: a CD (Method 1); a calibrated diffuse reflector (Method 2); or a quantum counter (Method 3). The latter
two methods use the instrument’s fluorescence detection system as a detector.
For Methods 1 and 2, the detector and diffuse reflector are calibrated for responsivity and reflectance, respectively, as a
function of wavelength. For Method 2, excitation and emission wavelength selectors are scanned synchronously, and the
spectral correction for the emission channel [see Signal Level (Relative Emission)] must be applied to the measured intensities.
Method 3 should be used only in the quantum counter’s effective wavelength range where a wavelength-independent response
can be achieved. Method 1 using a CD has fewer caveats than do the other two methods. A CD is placed in the sample position,
and the output is measured as a function of emission wavelength by scanning the excitation wavelength selector over the
excitation region of interest using the same instrument settings as those used with the sample. The known responsivity of the
CD is used to calculate the flux of the excitation beam. If the instrument’s reference detector is used to measure the intensity
of the excitation beam simultaneously with the CD, then the correction factor for the responsivity of the reference detector also
can be calculated.

Intensity and Sensitivity

The absolute value of the fluorescence signal measured by the detection system depends not only on the sample itself but
also on the excitation intensity of the sample and the optical geometry of the instrument. Therefore, determination of
instrument-independent fluorescence intensity of any sample or the absolute responsivity of any detection system in terms of
the intensity of the sample or measured by the detector, respectively, relative to the excitation intensity can be difficult.
The most accurate way to calibrate an instrument for absolute intensity is to use conventional standards-based methods such
as those that employ a calibrated light source or a calibrated detector in combination with a calibrated reflector. These methods
require user skill and knowledge. Also, the (typically annual) certification and recertification are expensive. In addition, these
standards tend to be bulky and are not compatible with many instruments. Thus, most researchers use simpler alternative
standards and methods.

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One approach is to correlate fluorescence signals to analyte concentrations using calibration curves or MESF units (see Analyte
Concentration—Calibration Curves). Another approach is to measure the intensity of a standard sample that can be expected to
always give the same fluorescence intensity under the same conditions.
Organic dyes, such as those used as fluorescent probes, generally are not good choices for intensity standards because of
issues with photobleaching, stability, and reproducible concentration. If organic dyes are used, then those with known high
purity and known shelf life, such as those produced by national metrology institutes, are recommended for single use (i.e.,
analysts should use a fresh solution for every measurement).
A better alternative is to use fluorescent samples that are stable over time even when exposed to light. For example,
fluorescent, inorganic glasses with well-characterized photostability and spectral properties and long shelf lives are commercially
available. Such materials can be used for determining a quasi-absolute intensity scale by measuring fluorescent signal at fixed
wavelength values within their recommended range using specified experimental parameters such as bandwidths, excitation
intensities, and temperatures.
The sensitivity of a fluorescence instrument is determined by measuring the signal-to-noise ratio of the fluorescence signal
of intensity standards. The Raman line of water often is used to measure sensitivity in a similar way, but the Raman signal typically
is strong enough only to be useful in the UV region. Analysts can use organic dye solutions to measure instrument sensitivity
or limits of detection with caveats that are identical to those that apply when the solutions are used as intensity standards.
The methods outlined here yield a quasi-absolute intensity scale that should be instrument independent for instruments with
similar optical geometries, designs, and settings. Results of these measurements enable comparison of the sensitivity of different
fluorescence instruments, but these comparisons should be approached with caution because of the relatively large and
difficult-to-quantify uncertainties involved.

PROCEDURE VALIDATION

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Validation of an analytical procedure using fluorescence demonstrates that the result is valid within a specified, acceptable
uncertainty budget. Instrument qualification, which also may involve instrument calibration, usually is part of the process, and
analysts also must consider sample-related errors (see Sample-based Factors). These can arise from concentration, anisotropy,
photostability, and shape of the sample, in combination with effects of the instrument’s optical geometry. All suspected errors
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should be quantified and combined to give a total estimated error that must be less than the method-specific, acceptable limit.

GLOSSARY
Absorptivity (a): A measure of the absorption of radiation from an incident beam as it traverses a sample, which is equal
to the quotient of:
ffi
A/bc

A = absorbance
b = path length (cm)
c = concentration (mg/mL)
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Also referred to as specific absorption coefficient by the International Union of Pure and Applied Chemistry.
Absorption coefficient (α): A measure of absorption of radiation from an incident beam as it traverses a sample according
to Bouguer’s Law:

I/I0 = e–ab

I = transmitted intensity
I 0 = incident intensity
e = base of natural logarithm
a = absorptivity
b = path length of the beam through the sample
Note that transmittance T = I/I0 and absorbance A = −log T.
Beer–Lambert law (or Beer’s law or Beer–Lambert–Bouguer law): In the absence of any other physical or chemical
factors, Aλ is proportional to path length, b, through which the radiation passes and to the concentration, c, of the substance
in solution in accordance with:

A λ = ελcb

ελ = molar absorptivity
c = solute concentration (M/L)
b = path length (cm)
Calibrated detector (CD): A light detector whose responsivity as a function of wavelength has been determined along
with corresponding uncertainties.
Calibrated light source (CS): A light source whose radiance as a function of wavelength has been determined along with
corresponding uncertainties.
Calibrated diffuse reflector (CR): A Lambertian reflector whose reflectance as a function of wavelength has been
determined along with corresponding uncertainties.

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Certified reference material (CRM): A material with properties of interest, the values and corresponding uncertainties
of which have been certified by a standardizing group or organization. A “reference material, accompanied by documentation
issued by an authoritative body and providing one or more specified property values with associated uncertainties and
traceabilities, using valid procedures” [International Vocabulary of Metrology (VIM) 5.14].
Diffuse scatterer: A material that scatters light in multiple directions. This includes diffuse reflectors, which often are
Lambertian, and scattering solutions, which are not Lambertian.
Fluorescence anisotropy (r): A measure of the degree of polarization of fluorescence:

r = (I|| − I⊥)/(I|| + 2I⊥)

I || = observed fluorescence intensity when the fluorometer’s emission polarizer is oriented parallel to the direction of the
polarized excitation
I ⊥ = observed fluorescence intensity when the fluorometer’s emission polarizer is oriented perpendicular to the direction
of the polarized excitation
Fluorescence band: A region of a fluorescence spectrum where the intensity passes through a maximum, usually
corresponding to a discrete electron transition.
Fluorescence lifetime:1 A parameter describing the time decay of the fluorescence intensity of a sample component. If a
sample decays by first-order kinetics, this is the time required for its fluorescence intensity and corresponding excited-state
population to decrease to 1/e of its initial value.
Fluorescence quantum efficiency: The ratio of the number of fluorescence photons leaving an emitter vs. the number
of photons absorbed.
Fluorescence quantum yield (Φ): The probability that a molecule or species will fluoresce once it has absorbed a photon.

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This quantity is an innate property of the species and typically is calculated for a sample as the ratio of the number of molecules
that fluoresce vs. the number of molecules that absorb. Fluorescence quantum yield values range from 0 (i.e., no molecules
fluoresce) to 1 (theoretical maximum in which all molecules that had absorbed radiation fluoresce).
Flux (or radiant flux): The rate of propagation of radiant energy, typically expressed in watts. Spectral flux is the flux per
unit spectral bandwidth, typically expressed in watts per nanometer.
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Grating equation: It describes the relationship between the angle of diffraction and the wavelength of radiation that is
incident on a grating:

mλ = d(sin α + sin β)

m = diffraction order
ffi
d = groove spacing on the grating
α = angle of the incident wavefront relative to the grating normal
β = angle of the diffracted wavefront relative to the grating normal
Inner filter effects: A decrease in the measured quantum efficiency of a sample caused by extensive absorption of the
excitation beam or reabsorption of the emission by the sample itself. This causes the measured quantum efficiency to depend
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on the absorbance, concentration, and excitation and emission path lengths of the sample.
Intensity: A measure of the amount of electromagnetic energy present. This general definition is synonymous with or
directly proportional to the signal output of a photodetector or the flux of a sample or light source. A more specific definition
often used in radiometry is: the radiant flux per unit solid angle from a point source, which typically is expressed as watts per
steradian (W/sr). [NOTE—Steradian corresponds to the SI unit of solid angle.]
Lambertian reflector: A surface that reflects light according to Lambert’s law, i.e., the light is unpolarized and has a
radiance that is isotropic or independent of viewing angle.
Limit of detection (LOD): An estimate of the lowest concentration of an analyte that can be measured with a given
procedure, often taken to be the analyte concentration with a measured signal-to-noise ratio of 3.
Noise level: The peak-to-peak noise of a blank.
Photobleaching: A loss of emission or absorption intensity by a sample caused by exposure to light. This loss can be
reversible or irreversible, and the latter typically is referred to as photodegradation or photodecomposition.
Quantum counter: A photoluminescent emitter with a quantum efficiency that is independent of excitation wavelength
over a defined spectral range. When a quantum counter is combined with a detector to give a response proportional to the
number of incident photons, the pair is called a quantum counter detector.
Quasi-absolute fluorescence intensity scale: A fluorescence intensity scale that has been normalized to the intensity
of a fluorescent reference sample or artifact under a fixed set of instrumental and experimental conditions. This artifact should
demonstrably yield a fluorescence intensity that is reproducible with time and between instruments under a fixed set of
conditions.
Raman scattering: The inelastic scattering of radiation (the wavelengths of the scattered and incident radiation are not
equal) by a sample that occurs because of changes in the polarizability of the relevant bonds of a sample during a molecular
vibration. Unlike fluorescence, the radiation being scattered is not required to be in resonance with electronic transitions in the
sample.
Rayleigh scattering: The elastic scattering of radiation by a sample; i.e., the scattered radiation has the same energy (same
wavelength) as the incident radiation.
Responsivity (spectral): The ratio of the photocurrent output and the radiant power collected by a light-detection system.
Spectral responsivity is the responsivity per unit spectral bandwidth.
Sensitivity: A measure of an instrument’s ability to detect an analyte under a particular set of conditions.
1 Boens
N, Qin W, Basaric N, et al., Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy. Anal Chem. 2007;79(5):
2137–2149.

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Spectral bandwidth (or spectral bandpass or resolution): A measure of the capability of a spectrometer to separate
radiation or resolve spectral peaks of similar wavelengths. Usually observed as the triangular dispersion of an emission line, this
parameter is taken to be the full width at half the peak maximum (FWHM).
Spectral slit width: The mechanical width of the exit slit of a spectrometer divided by the linear dispersion in the exit slit
plane. In practice, observed as the triangular dispersion of an emission line, this width includes all the transmitted wavelengths
for a given slit setting.
Transition dipole moment: An oscillating dipole moment induced in a molecular species by an electromagnetic wave
that is resonant with an energy transition of the species, e.g., an electronic transition. Its direction defines the transition
polarization, and its square determines the intensity of the transition.

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