REVIEW

Pharmaceutical Applications of Separation of Absorption and

Scattering in Near-Infrared Spectroscopy (NIRS)
ZHENQI SHI, CARL A. ANDERSON
Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282

Received 14 December 2009; accepted 20 April 2010

Published online 2 June 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22228

ABSTRACT: The number of near-infrared (NIR) spectroscopic applications in the pharmaceu-

tical sciences has grown significantly in the last decade. Despite its widespread application, the
fundamental interaction between NIR radiation and pharmaceutical materials is often not
mechanistically well understood. Separation of absorption and scattering in near-infrared
spectroscopy (NIRS) is intended to extract absorption and scattering spectra (i.e., absorption
and reduced scattering coefficients) from reflectance/transmittance NIR measurements. The
purpose of the separation is twofold: (1) to enhance the understanding of the individual roles
played by absorption and scattering in NIRS and (2) to apply the separated absorption and
scattering spectra for practical spectroscopic analyses. This review paper surveys the multiple
techniques reported to date on the separation of NIR absorption and scattering within phar-
maceutical applications, focusing on the instrumentations, mathematical approaches used to
separate absorption and scattering and related pharmaceutical applications. This literature
review is expected to enhance the understanding and thereby the utility of NIRS in pharma-
ceutical science. Further, the measurement and subsequent understanding of the separation of
absorption and scattering is expected to increase not only the number of NIRS applications, but
also their robustness. ß 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci
99:4766–4783, 2010
Keywords: near-infrared spectroscopy; absorption spectroscopy; light-scattering; Monte
Carlo; radiative transport equation

INTRODUCTION the sample matrix, multivariate modeling is often

The number of near-infrared spectroscopic applica-
tions in pharmaceutical science has grown signifi-
cantly in the last decade. These applications have
rapidly permeated the research and development
activities in the pharmaceutical industry, including
raw material characterization, powder blending
monitoring, granulation process control, tablet
manufacture and finished products characteriza-
tion.1,2 Much of the appeal of this technique is due to
the fact that a wealth of chemical and physical
information can be obtained noninvasively within
seconds, often without the need for any sample
preparation.
As NIR spectra contain information pertaining
to both the chemical and physical properties of
Correspondence to: Carl A. Anderson (Telephone: 412-396-1102;
Fax: 412-396-4660; E-mail: andersonca@duq.edu)
Journal of Pharmaceutical Sciences, Vol. 99, 4766–4783 (2010)
ß 2010 Wiley-Liss, Inc. and the American Pharmacists Association
required to correlate spectral information with the
chemical or physical properties of the analyte of
interest. In spite of the extensive application of NIRS,
the underlying optical behavior of NIR radiation in
pharmaceutical materials is often not mechanisti-
cally well understood.
It is well known that two primary events occur
when NIR light impinges on a turbid medium (e.g.,
biological tissues or pharmaceutical solids): absorp-
tion and scattering.2 Absorption reduces the intensity
of photons of specific energy due to an alteration of
the molecular dipole of a bond; therefore, chemical
attributes such as concentration are expected to affect
absorption events. Scattering, on the other hand, is
caused by mismatched refractive indices at particle–
air/particle–particle interstitial spaces within the
sample; therefore, physical parameters such as
sample density and porosity are dominant factors
in determining scattering events. Considerable suc-
cess has been achieved in the field of biomedical optics
when separated absorption and scattering properties

4766 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
 SEPARATION OF ABSORPTION AND SCATTERING IN NIRS 4767

were used to describe underlying optical behaviors in THEORY—LIGHT PROPAGATION IN TURBID

tissue samples.3,4 The separated absorption and MEDIA
scattering properties are often used to understand
the optical behaviors in tissues, correlating them Turbid Medium
directly with the chemical and physical features of the
In physics and chemistry, light propagation is often
tissue medium. The major goal in tissue optics is to
described by its wave-like and particle-like proper-
utilize the separated absorbing and scattering proper-
ties. Wave-like properties indicate light to be an
ties of tissue samples for diagnostic and therapeutic
oscillating electromagnetic (EM) field with a con-
applications.
tinuous range of energies. This is also referred to as
Due to the common features shared by tissue
the classical theory of light propagation. Particle-like
samples and pharmaceutical solids (as both are
properties indicate that light waves consist of
turbid media), more and more attention has been
packets of energy called photons, which are described
paid to the separation of NIR absorption and
by the quantum model. Quantum theory introduces
scattering in pharmaceutical applications. The two
the idea that light and matter exchange energy as
main purposes of the separation are to improve the
photons.
understanding of individual roles played by absorp-
It is well known that light propagation in dilute,
tion and scattering in NIRS, and to utilize the
nonscattering solution systems is described by the
separated absorption and scattering spectra for
Lambert–Beer’s law. Since a diluted sample does
qualitative and quantitative applications. To date,
not scatter light, a fixed path-length is typically
five techniques to separate NIR absorption and
used in Lambert–Beer’s law. If the sample multiply
scattering of pharmaceutical related materials have
scatters light, then a distribution of path-lengths
been reported: spatially resolved spectroscopy,5–8
will be observed. Multiply scattering, also called
frequency-resolved spectroscopy,9–21 time-resolved
‘‘multiple scattering,’’ is the photon behavior in
spectroscopy,22–27 the integrating sphere based
which individual photons are scattered a large
reflectance and transmittance measurements,28–31
number of times before eventually escaping from or
and measurements of remission, absorption and
being absorbed by the medium. Thus, the descrip-
transmission fractions through layers of material
tion of light propagation in strongly scattering
of different thicknesses.32,33 A review of how these
media becomes more complicated than is expressed
techniques are applied specifically to pharmaceutical
by the Lambert–Beer’s law. Examples for multiply
application has yet to be reported.
scattering media are concentrated solutions, col-
A clear understanding of these optical phenomena
loids, semi-solids, and solids, etc. This is also the
is expected to facilitate the application of NIRS to
reason that models for light propagation in strongly
pharmaceutical science, particularly as efforts to
scattering media have been used so widely in
engage process analytical technology (PAT)-based
medicine (i.e., tissue optics), agriculture, the paper
manufacturing strategies intensify. Therefore, a
industry, and pharmaceuticals. The most common
literature review detailing the separation of absorp-
examples of strongly scattering media in the
tion and scattering in pharmaceutical materials is
pharmaceutical field are solid materials, either in
expected to aid in the implementation of NIRS
free powder or consolidated compact forms. A
through a fundamental understanding of the under-
sampling medium exhibiting the multiply scatter-
lying optical phenomena. This fundamental under-
ing property is normally referred to as a turbid
standing will greatly increase the potential success
medium.
and longevity of NIRS methods in the pharmaceutical
industry.
Radiative Transport Equation
The goal of this paper is to review the approaches
that have been applied in pharmaceutical analyses, Considerable success has been achieved in describ-
focusing on the following perspectives: (1) instru- ing the light propagation in turbid media by the
mentation, (2) mathematical approaches to separate application of radiative transfer theory (radiative
absorption and scattering, and (3) related pharma- transport equation, RTE).3,4 Radiative transfer
ceutical applications for the individual techniques theory is not specific to light and has other
as mentioned above. The review begins with a important applications in areas such as neutron
theoretical background of light propagation in transport and thermodynamics. In the RTE formal-
turbid media, followed by a literature survey and a ism, light propagation is considered equivalent to
comparison among these techniques from both the flow of discrete photons, which are either
theoretical and application standpoints, and con- absorbed or scattered by the medium. RTE only
cludes with a discussion of the authors’ perspective accounts for the transport of light energy in the
on future trends of these techniques in the pharma- medium. It ignores the wave amplitude and
ceutical applications. phases and does not itself include effects such as

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
4768 SHI AND ANDERSON

diffraction, interference, or polarization. Further, Diffusion Approximation to the RTE

no correlation between the radiation fields is
Due to the multiply scattering events that occur when
considered in RTE (i.e., photons are independent
NIR light interacts with turbid media, the diffusion
of each other).
approximation is commonly used to simplify the RTE.
In radiative transfer theory, a small packet of light
The diffusion approximation is applicable to situations
energy (I) is considered by defining its position (r)
where scattering dominates the light propagation
directed in a cone of solid angle (dV) and oriented in
process. In the diffusion process, the particles (in the
the direction of propagation ð^ sÞ in a sample medium.
current case, photons) move through a medium in a
As it propagates in the medium, the packet loses a
series of steps of random length and direction (i.e.,
fraction of its energy due to absorption and scattering
random walk). Each step begins with a scattering
out of s^ (the first term on the right side of Eq. 1), but
event that is equally likely to be taken in any direction.
also gains energy from light scattered into the s^
In the diffusion approximation, the scattering
direction from other s^0 directions (the second term on
event in RTE is described by the reduced scattering
the right side of Eq. 1). These processes are quantified
coefficient m0s , which is related to the previously
by the integral-differential equation known as the
defined parameters by
RTE:3
~ m0s ¼ ð1  gÞms (4)
s^  rIðr; s^Þ ¼ ðma þ ms ÞIðr; s^Þ
Z
m þ ms where g is the anisotropy factor, describing the
þ a pð^s  s^0 ÞIðr; s^ÞdV ð1Þ
4p angular distribution of SPF between s^0 and s^ in
Eq. (1). The anisotropy factor is zero for isotropic
where pð^ s  s^0 Þ is the scattering phase function (SPF)
scattering. A nonzero g value is representative of
between the s^ and s^0 directions. ma and ms are the
anisotropic scattering, in which total forward scatter-
absorption and scattering coefficients of the sample
ing is described by g ¼ 1, while total backward
medium, respectively. In RTE, the ms and ma are
scattering is described by g ¼ 1. As it can be seen
defined as follows.
in Figure 1, a scatterer with a g in the range of 0 to 1
A medium containing a uniform distribution of
means it is more likely to forward-scatter the incident
identical scattering and absorbing particles is char-
photons, while a scatter with g ranging from 1 to 0
acterized by the scattering and absorption coeffi-
indicates it is more likely to backward-scatter the
cients, respectively
incident photons. Pharmaceutical solids are reported
ms ¼ rs s (2) to demonstrate substantial forward scattering within
the NIR wavelength region.2
ma ¼ rs a (3) After introducing m0s , the light propagation beha-
vior in a sample with optical properties ma, ms, and
where ss and sa are the cross sectional areas of the g 6¼ 0 can be alternatively described by the same
scattering and absorption particles, respectively, sample with optical properties ma, m0s , and g ¼ 0.
which describe the propensity to scatter and absorb. Alternatively stated, the reflectance and transmit-
r is the number density of scattering and absorption
particles in the sample medium. The standard units
of for ms and ma are cm1.
Different terminologies are often used to represent
the ms and ma when individual techniques are used to
separate absorption and scattering, despite the fact
that they often share the same underlying optical
definitions. The absorption coefficient is defined as
the probability of photons being absorbed, while the
scattering coefficient is defined as the probability
of photons being scattered. The reciprocal of ms is
the scattering mean free path, representing the
average distance a photon travels between consecu-
tive scattering events. The reciprocal of ma is the
absorption mean free path, representing the average
distance a photon travels between consecutive
absorption events. Absorption and scattering coeffi-
cients together are often referred to as the optical Figure 1. The angular distribution of SPF for g ¼ 0.9,
coefficients or the optical properties of a sample 0.5, 0.1, 0.2, 0.5, and 0.8, assuming the light impinges
medium. upon the center of the polar plot from the left.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010 DOI 10.1002/jps

tance for a sample with optical properties ma, ms, and
g 6¼ 0 are the same as those for the same sample with
optical properties ma, m0s , and g ¼ 0. This is referred to
as the ‘‘Similarity Principle’’ in tissue optics.34
Therefore, the optical properties needed to describe
light propagation behavior in turbid media are
simplified to ma and m0s .
Based on the first-order expansion in the unit
vector s^, the diffusion equation is obtained as follows;
the detailed derivation can be found elsewhere.3,35,36
@ tion.
fðr; tÞ ¼ Dr2 fðr; tÞ  ma cm fðr; tÞ þ Qðr; tÞ
@t
In the diffusion approximation, the properties of
photon movement in the medium are contained in the
diffusion constant, D ¼ ð1=3½ma þ m0s Þ. fðr; tÞ is the
fluence rate, which is the light intensity per unit area
at position (r) at a given time (t). F(r, t) is the net
intensity vector (i.e., diffuse photon flux), which is the
photon energy per unit area in the direction of s^. cm
symbolizes the speed of light in the sample medium.
In order for the diffusion approximation to the RTE
to describe the light propagation behaviors in turbid
media, the following assumptions are typically
considered.
SEPARATION OF ABSORPTION AND SCATTERING IN NIRS 4769
complex illumination geometries. These situations
typically require assumptions regarding individual
boundary conditions and parameters to generate a
closed form analytical solution to the RTE (Eq. 1).
Thus, analytical solutions for realistic scenarios are
complicated at best (if an analytical solution even
exists). For cases when the analytical solution is not
available, the problem can be approached using
numerical techniques. The most widely used
approach for radiative transfer theory is Monte
Carlo (MC) simulation method-based photon migra-
(5)
Monte Carlo refers to the city in Monaco, where
the primary attractions are casinos that offer games
of chance. The random behavior in games of chance
is similar to how MC simulation statistically
samples the probability distribution for the photon
migration parameters, such as step size, scattering
direction, internal reflection/out of boundary, etc.
These parameters are simulated using functions of
random number generators. Because of these func-
tions, individual photon movement can be traced
step-by-step through the sample medium, and the
distribution of light (i.e., reflectance and/or trans-
mittance) in the system can be recorded from these
individual photon trajectories. As the number of
photons in the simulation grows toward infinity, the
 m0s =ðma þ m0s Þ, also known as the albedo (v0), is
close to unity, that is, when the absorption of the
medium is low. If scattering is not dominant, the
photon migration behavior cannot be appropri-
ately described by the random walk. Then the
diffusion approximation to RTE also does not
stand.37
MC simulation for the light distribution approaches
an analytical solution to the RTE. The actual
number of photons necessary for a realistic result
depends on the specifics of the simulation and which
quantities one desires to determine. As few as 3000
photons may be adequate for determining diffuse
reflectance from a sample, while more than 100000
 The point of interest is far from sources or bound-
aries. A common criterion is the distance
between the light source and detector is larger
than 10 times the mean free path of the photons
in the sample. This indicates that the diffusion
approximation to RTE is only suitable for
describing photon migration behaviors that
experience large numbers of scattering events
with small scattering angles, in comparison with
photons experiencing a few scattering events
with large scattering angles.22
may be required for a complex three-dimensional
simulation.3
There are several advantages to using the MC
method to describe photon migration behavior.3,4
First, the initial large-scale applications of MC
methods were radiative transfer problems involving
neutron transport. Thus, the MC approach is well
suited for problems involving light transport in turbid
media because a photon can be treated as a neutron
particle whose propagation behaves according to the
rules of radiative transport. Second, the algorithms
 The sample medium has to be of finite thickness.
If the sample becomes thinner to the point where
its thickness is comparable to theffi effective pene-
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
tration depth 1= 3ma ðma þ m0s Þ, the diffusion
approximation will break down as a result of
the increased relative importance of potential
photon–photon interactions.34
Monte Carlo simulation
Many situations of practical interest involve a
variety of light sources, multiple sample types, and
DOI 10.1002/jps
for implementing the basic elements of an MC
simulation are straightforward. Third, the technique
possesses a great deal of flexibility and is widely
applicable to practical transport problems. Through
the use of corresponding mathematical relationships,
MC simulation has the ability to simulate virtually
any source, detector, and sample boundary condition,
as well as any combination of sample optical proper-
ties. Finally, the MC approach can be used for any
albedo and SPF.
A sample MC protocol for simulation-based photon
migration is illustrated in Figure 2.38
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
4770 SHI AND ANDERSON
Figure 2. Schematic diagram of Monte Carlo simulation.
The figure was adapted from Wang et al.38
TECHNIQUES USED FOR SEPARATING
ABSORPTION AND SCATTERING IN NIRS
Integrating Sphere-Based Reflectance and
Transmittance Measurement
The integrating sphere-based approach, reported by
Fricke and coworkers is the first documented method
for the separation of NIR absorption and scattering in
pharmaceutical samples.28–31 Measurement of dif-
fuse reflectance (Rd) and diffuse transmittance (Td)
provides access for deconvolution of analytical
equations to determine ma and m0s .
Instrumentation
Integrating sphere-based approaches can be gener-
ally classified into two different instrument set-ups.34
The first set-up uses a single integrating sphere for
both the reflectance and transmittance geometries
(Fig. 3A). The incident light can be either a collimated
beam or diffuse irradiation. The measurement via
diffuse irradiation is less accurate than that made
with collimated irradiation as a result of the high
background signal of the diffuse source in the
reflectance measurement.34
The other set-up consists of a double integrating
sphere system (Fig. 3B), where the sample is placed in
a common port between two spheres. One of the main
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
Figure 3. Integrating sphere based reflectance and
transmittance measurements. (A) Single sphere measure-
ment. (B) Double sphere measurement. The figure was
adapted from Wilson.34
advantages of using a double sphere is to increase
signal in both spheres over the single-sphere case,
which is a result of the ‘‘cross talk’’ between the two
spheres.34 Additionally, the double sphere provides
the opportunity to determine Rd and Td simulta-
neously. This instrument configuration has not yet
been applied to pharmaceutical samples.
In the integrating sphere-based approach, the
measurement is designed to obtain values of both
Rd and Td. Therefore, the sample must have a finite
thickness, which is dependent on the optical proper-
ties of the sample. If the sample is too thick, not
enough signal will be detected in Td. If the sample is
too thin, however, the assumption of the diffusion
approximation will be violated as a result of
individual photons interfering with each other.34
It has been suggested that performing a complete
calibration of the experimental set-up using samples
of known absorption and scattering properties over
the range of those anticipated for samples and
wavelengths of interest is essential to obtain accurate
measurements of Td and Rd.34 Thus, both single-
sphere and double-sphere systems require calibration
using standard samples to characterize the method
accuracy (i.e., percentage deviation from known
values of the standards) in order to further correct
the estimated ma and m0s .
Mathematical Approaches to Determine la and ls0
Fricke and coworkers were the first to develop the
closed-form equations for Rd and Td, and they used
these equations to determine the optical properties of
pharmaceutical samples.28–31 The detailed deriva-
tions can be found elsewhere.30 Briefly, a three-flux
approximation was applied to derive the measured
DOI 10.1002/jps
 SEPARATION OF ABSORPTION AND SCATTERING IN NIRS 4771

quantities of transmission (Td) and reflectance (Rd). pharmaceutical powder samples within the infrared
The three-flux approximation represents the three (IR) and NIR ranges.28,29,31
surfaces through which light propagates: transmit- The first paper addressed the general information
tance, reflectance, and side scatter (as described in of absorption and scattering properties of pharma-
Mathematical Approaches to Determine ma and m0s ceutical powders in the IR range,29 which was later
Section). confirmed in the NIR range.31 The inter-relationship
  between the absorption and reduced scattering
Td v 0 ; t 0 coefficients for specific materials was investigated.
 
cg aT kt 0 bT kt 0 t0
It was found that an inverse correlation between the
¼ e þ e þ 3e TF reduced scattering and absorption coefficient existed
F 1  ð2=3Þk 1 þ ð2=3Þk

for the sample materials (e.g., lactose and micro-
þ et0 TF crystalline cellulose powder), in which a peak in the
(6) absorption coefficient always corresponded to a valley
in the reduced scattering coefficient. The inverse
 
Rd v 0 ; t 0 relationship between the absorption and reduced
  scattering coefficients was also confirmed using a
cg aR bR 3
theoretical Mie calculation.29
¼ þ þ þ 3e2to RF
F 1 þ ð2=3Þk 1  ð2=3Þk 5 The wavelength dependences of the optical coeffi-

þ e2t0 RF cients were also explored. The reduced scattering
coefficient gradually decreased with increasing wave-
(7) lengths (and absorption). Further, the absorption
The formulae for calculating constants c, g, k, aT, bT, coefficients showed wavelength dependence due to
aR, bR, A, and B in Eqs. (6) and (7) can be found specific absorption bands within certain wavelength
elsewhere.30 In addition, F, TF, and RF are experi- ranges. The wavelength dependence of the reduced
mental constants, representative of the impinging scattering coefficient was also found to be material
flux of the light source, the transmission and dependent. The reduced scattering coefficients of
reflectance coefficient of the supporting layer on lactose and microcrystalline cellulose powder were
which the Td and Rd measurements of the powder found to vary over the observed wavelength range,
sample were measured. Except for F, TF, and RF, the while the reduced scattering coefficients of paraceta-
rest of constants shown in the right side of Eqs. (6) mol and ascorbic acid powder were relatively
and (7) are functions of the scaled albedo ðv 0 Þ and the constant. Given the wavelength dependency of
scaled optical depth (t ).30 The v 0 and t are scattering, Fricke and coworkers pointed out the
defined as potential invalidity in the commonly used scattering
correction methods, such as multiplicative scattering
v0 ð1  gÞ correction (MSC) and standard normal variate (SNV),
v 0 ¼ (8)
1  v0 g which assume constant scattering across the wave-
length axis.
t ¼ tð1  v0 gÞ (9) Additionally, the authors investigated the effect of
particle size on the optical coefficients. It was
where t ¼ m0t x, m0t ¼ m0s þ ma , and x represents the
demonstrated that the reduced scattering coefficient
photon position along the depth-axis of the sample.
was inversely related to particle size in both the IR
Since Eqs. (6) and (7) express the diffuse reflectance
and NIR ranges; smaller particle size led to a larger
and transmittance as a nonlinear function of v 0 and
reduced scattering coefficient. Meanwhile, the
t , nonlinear regression tools (e.g., Newton–Raphson
absorption coefficient was also found to increase
method) are typically applied to fit the measured Rd
when particle size was reduced, especially within the
and Td profiles in order to estimate the v 0 and t .
IR range where strong absorption bands are
Subsequently, the reduced scattering coefficient m0s
located.29 The correlation between the absorption
and absorption coefficient ma can be determined using
coefficient and particle size diminished in the NIR
Eqs. (8) and (9).29 Since the determined optical
range, except for large particle size difference, such as
coefficients are typically normalized by the sample
that observed for 90 mm versus 490 mm ascorbic acid
density, the resultant units for the optical coefficients
powder.31 Fricke and coworkers attributed the
determined by this approach are m2/kg.
relationship between the absorption coefficient and
particle size to the decreased hidden mass in the small
Related Pharmaceutical Applications
particles, which results in a corresponding increase in
Fricke and coworkers conducted a series of studies absorption. The hidden mass was defined as the
in which they utilized the integrating sphere- portion of material contributing to the total mass
based approach to determine optical coefficients of but not contributing to absorption. For example,

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
4772 SHI AND ANDERSON
absorption is much stronger for several small
particles having the same mass as a single large
particle, which is a consequence of the decreased
hidden mass of the former.29,31
Given the effects of particle size on the optical
coefficients, especially for the absorption coefficient,
Fricke and coworkers realized its potential impact on
the quantitative analysis of powder mixtures.29 The
authors cautioned that the development of a proper
calibration for a two-component mixture, subsequent
spectral analysis of unknown mixtures may contain
significant errors. They postulated that this might be
due to a change in the degree of agglomeration (owing
to humidity), which alters the particle size distribu-
tion of the mixture and leads to differences in the
absorption intensity and both optical coefficients.29
However, the authors did not address any potential
solutions to the variability in the absorption coeffi-
cient as a result of physical variation (e.g., particle
size) and its potential effect on prediction errors in
future samples.
The same group of researchers applied the
extracted absorption and reduced scattering coeffi-
cients to enhance the prediction capability of a
method for determining the amount of active
ingredient in two-component mixtures containing
paracetamol and lactose.28 Since MSC treats scatter-
ing as a wavelength-independent phenomenon, MSC
was used only on wavelength regions that were
independent of scattering (i.e., wavelength ranges for
which constant reduced scattering coefficients of the
powder mixture were observed). Partial least squares
modeling resulted in a more accurate (lower RMSEC)
model compared to MSC applied to the entire
wavelength region. Additionally, an artificial neural
network (ANN) was trained to build a calibration
model between separated absorption coefficients and
paracetamol concentrations. The ANN-derived model
showed significantly lower RMSEC and RMSEP
compared to the PLS calibration. Burger et al.
attributed the enhanced performance of the ANN to
its capacity to model nonlinear relations. The
application of an ANN was justifiable as a certain
degree of nonlinearity was found between absorption
coefficient and paracetamol concentration.
Measurements of Remission, Absorption, and
Transmission Fractions through Layers of Material of
Different Thicknesses
Separation of absorption and scattering was reported
for diffuse reflectance measurements of pharmaceu-
tical samples of different thicknesses.32,33 An inte-
grating sphere is typically used to acquire the
measurement. The difference compared to the
approach used in the section of Integrating Sphere-
Based Reflectance and Transmittance Measurement
is that here, only reflectance is used; no transmittance
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
component is involved. Instead of estimating the
absorption and reduced scattering coefficients, a
simplified solution of the Kubelka–Munk (K–M)
function describing light flux into and from samples
was used to calculate the K–M absorption (K) and
scattering (S) coefficients. The following equations
were used to describe the relation between S and K
within diffuse reflectance measurements.
 
2:303 R1 R1 ð1  R1 R0 Þ
S¼ log (10)
d 1  R21 R1  R0
 
2:303 1  R1 R1 ð1  R1 R0 Þ
K¼ log (11)
2d 1 þ R1 R 1  R0
Here, R0 denotes the spectrum measured at a defined
sample thickness (d) and R1 denotes the measure-
ment of the same sample with an optically infinite
thickness. As it can be seen from Eqs. (10) and (11), K
and S can be directly calculated from the reflectance
measurements.
Although the coefficients K and S used in K–M
theory are not directly comparable with ma and m0s ,
they are related through the relationship expressed
in Eq. (12).17,21,29
  
K 8 ma
¼ (12)
S 3 m0s
The sole pharmaceutical application of the optical
coefficients determined by this method was reported
in terms of hard model constraints for multivariate
curve resolution.33 Multivariate Curve Resolution is a
group of chemometric algorithms that help resolve
mixtures by determining the number of constituents,
their spectral profiles and their estimated concentra-
tions when no prior information is available about the
nature and composition of these mixtures. The
dataset contained NIR spectra of pharmaceutical
tablets compressed at 31, 156, and 281 MPa. The
spectra of the tablets compressed at 31 MPa were
used for calibration, while the remaining spectra
were reserved for validation. It was found that
multivariate curve resolution-alternating least
squares (MCR-ALS), using the background informa-
tion of K–M scattering and absorption coefficients,
was comparable in calibration, but superior in
validation, when compared to PLS modeling without
any spectral pretreatment. The study also showed
slightly better validation performance for optical
coefficients-based MCR-ALS, compared to pure com-
ponent spectra based MCR-ALS and PLS modeling on
extended multiplicative scattering correction (EMSC)
preprocessed spectra. Moreover, only three samples
were necessary for a reliable and robust calibration
when optical coefficients-based MCR-ALS was
applied, despite the fact that strong changes in the
scattering behavior were expected.
DOI 10.1002/jps
 SEPARATION OF ABSORPTION AND SCATTERING IN NIRS
Time-Resolved Spectroscopy (TRS)
Absorption and scattering properties of a turbid
medium can be estimated by measuring the temporal
dispersion of a short light pulse as it propagates
through the medium. Such measurements have long
been of interest in atmospheric research. For
example, Weinman and Shipley39 used the time
dependence of a transmitted pulse to deduce the
optical thickness of clouds. This method was first
developed for medical applications, but it has since
been extended to other fields, such as pharmaceutics
and agriculture. It uses picoseconds laser pulses to
irradiate a sample. The light signal diffusively
remitted by the sample at a given distance from
the irradiation point is then temporally recorded. The
temporal shape of the pulse is altered by absorption
and scattering events as it passes through the sample.
By analyzing the modified temporal shape of the
pulse, the optical properties of that sample can be
deduced.
Instrumentation
The basic instrument set-up of TRS requires a
picoseconds laser pulse, a sample interface (either
reflectance or transmittance), and a photon-counting
system to record the temporal spreading. The most
recent instrument set-up was developed by the Lund
Institute of Technology, Sweden.24 This system
utilizes an index-guided crystal fiber for light delivery
and a streak camera, which is necessary to achieve
the temporal resolution. The optic arrangement of
this system is presented in Figure 4.24 The laser pulse
is focused into a 100 cm long index-guiding crystal
Figure 4. Optical arrangement of TRS. The figure was reproduced, with permission,
from Abrahamsson et al.24
DOI 10.1002/jps
4773
fiber (ICF). Due to the optical behaviors of an ICF,
light pulses with approximately the same temporal
width as the laser are accessible with a spectral width
spanning from 500 nm to at least 1200 nm. In contrast
to a single photon counting system, this system uses a
streak camera to provide a unique combination of a
relatively short acquisition time with high spectral
and temporal resolution. The system measures a
700 nm wavelength region with a spectral resolution
of 5 nm. The system has a total temporal range of
2.1 ns with resolution of 4.5 ps.
Since time resolution must be on the order of tens of
picoseconds, the major limitation for the current TRS
system is the expensive instrument set-up, including
both the pulsed laser and the photon-counting
detector.34 Also, the system is limited by its current
wavelength range, which is relatively narrow com-
pared to that of the NIR region.23 The primary reason
for this limitation is the lack of commercially
available efficient photon cathode materials for
streak tubes that operate in the NIR range.
Mathematical Approaches to Determine la and ls0
The analytical equations for TRS were initially
derived from tissue optics. It was developed by
Patterson et al.40 for either reflectance measurements
of a semi-infinite homogenous medium or reflectance/
transmittance measurements of a finite medium. To
date, all reported pharmaceutical applications of TRS
involved finite media.
After using specific boundary conditions on the
diffusion approximation to the RTE (Eq. 5), the
expressions for the reflectance R(d, t) and transmit-
tance T(d, t) at a specific time (t) for a sample with
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
4774 SHI AND ANDERSON

finite thickness (d), such as a pharmaceutical tablet, spreading and an assumed refractive index, the
are degree of scattering within the tablet matrix, in
terms of the total optical path length, was determined
Rðd; tÞ ¼ ð4pDcÞ1=2 t3=2 expðma ctÞ
( " # to be 20–25 cm. This indicated that very strong
 
z20 ð2d  z0 Þ2 multiple scattering events took place within the
 z0 exp   ð2d  z0 Þexp  sample. Monte Carlo simulation and a corresponding
4Dct 4Dct
" #) comparison with the experimental data estimated the
ð2d þ z0 Þ2 reduced scattering coefficients of the tablet to be on
þ ð2d þ z0 Þ exp  ð13Þ
4Dct the order of 500 cm1 at 790 nm.
The same group of researchers published a second
Tðd; tÞ ¼ ð4pDcÞ1=2 t3=2 expðma ctÞ paper focusing on the application of extracted reduced
( " # scattering coefficients from TRS to enhance the
ðd  z0 Þ2 quantitative analysis of pharmaceutical tablets
 ðd  z0 Þexp   ðd þ z0 Þ
4Dct through scatter correction.22 Pharmaceutical tablets
" # " # produced at different compression forces and various
ðd þ z0 Þ2 ð3d  z0 Þ2 granule sizes were used. Multiple comparisons were
 exp  þ ð3d  z0 Þ exp 
4Dct 4Dct performed between the scattering-corrected spectra
" #) and raw NIR spectra using different calibration and
ð3d þ z0 Þ2
 ð3d þ z0 Þexp  ð14Þ validation datasets. When compared to raw NIR
4Dct scans, the scatter-corrected spectra resulted in lower
RMSEPs across all of the evaluated conditions.
where c represents the speed of light propagation in
In addition, Abrahamsson et al.27 utilized the slope
the sample medium, D stands for the diffusion
of the time dispersion curve from the time-resolved
constant, and z0 ¼ ½ð1  gÞms 1 .
measurement to determine the chemical concentration
As can be seen in Eqs. (13) and (14), measurement of
of binary compacts containing iron oxide and micro-
either reflectance or transmittance allows for a direct
crystalline cellulose. When compared to traditional
estimate of ma and m0s . In order to deconvolve these
transmission-based NIRS, the time-resolved measure-
two equations and extract the optical coefficients,
ment resulted in a fivefold increase in accuracy for the
multiple methods have been reported, including non-
determination of iron oxide concentration. Further,
linear regression (Levenberg-Marquardt algorithm,
due to the direct relationship between the slope of time
LMA),22,24 least-square support vector machine (LS-
dispersion curve and light absorption, the calibration
SVM)26 and other linear approaches, that is, MAx-
model based on the time-resolved measurement
imum Determination for Solving Time-REsolved
reliably predicted the concentration of iron oxide
Spectroscopy Signal (MADSTRESS).25 Nonlinear
in samples with physical properties outside those
regression is the most commonly used method among
included in the calibration set.
pharmaceutical applications.22,24 In mathematics and
computing, LMA generates a numerical solution to a Frequency-Resolved Spectroscopy (Frequency Domain
problem by minimizing a function, which generally Photon Migration, FDPM)
nonlinear, over a space of parameters specific to
The principle of FDPM involves monitoring the time-
function. LMA can also be used as a (nonlinear) least-
dependent propagation characteristics of multiply
squares curve-fitting algorithm. Abrahamsson et al.
scattered light in turbid media. Briefly, this technique
applied LMA to determine the optical coefficients of
launches intensity-modulated light onto a multiply
phantom samples and compared this method to the
scattering medium via a single point source, and
measurements acquired using integrating sphere-
detects it at other discrete points of known distances
based approaches. A phantom sample is a type of lipid
from the incident light. Upon modulating the incident
emulsion (Intralipid, Sigma–Aldrich, St. Louis, MO)
light at various modulation frequencies or varying the
with known optical properties in the short NIR
source-to-detector distance, the measurements of
wavelength range. Phantom samples have been widely
phase-shift and amplitude attenuation can be deter-
used as standards in tissue optics to characterize
mined as functions of the optical properties of the
method accuracy. Abrahamsson et al.24 concluded that
sample medium. The propagation of such a photon
the two methods offered comparable results.
density wave within a turbid medium is influenced by
its absorption and scattering properties and can be
Related Pharmaceutical Applications
modeled by the diffusion approximation to the RTE. By
The first application of TRS to pharmaceutical solving the diffusion equation with appropriate
samples was published by Johansson et al.23 Trans- boundary conditions, the measurement data (i.e., the
mittance mode was used to acquire measurements of phase-shift and amplitude attenuation) can be used to
a 3.5 mm-thick tablet. Based on the temporal determine the optical properties of the sample medium

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010 DOI 10.1002/jps
 SEPARATION OF ABSORPTION AND SCATTERING IN NIRS
Figure 5. Schematic diagram of the FDPM setup with the enlarged insert denoting a
powder configuration for multiple scattering of photons. The figure was reproduced, with
permission, from Pan and Sevick-Muraca.12
(i.e., the absorption and reduced scattering coeffi-
cients).
Instrumentation
The latest instrument used in FDPM was developed
by Sevick-Muraca et al. This group holds a number of
patents on FDPM instrumentation and their related
applications.13–15
As shown in Figure 5,12 modulated light of
modulation frequency v (typically 30–200 MHz) is
launched from a monochromatic laser diode, which is
directed to a beam splitter to form reference and
sampling beams. The reference beam is delivered to
a reference photomultiplier tube (PMT) through a
1-mm diameter optical fiber. The sampling beam is
introduced to the sampling medium through a second
1-mm diameter optical fiber whose end is placed
within the sampling medium. A third 1-mm-diameter
fiber is located a distance of r from the point of
illumination to detect the propagated light. The
relative distance between the source and detector
fibers has to be at least 10 times that of the scattering
mean free path to ensure multiple light scattering,
typically 1–15 cm. The source and detector fibers are
normally maintained in a coplanar geometry. Detec-
tion is accomplished with a second PMT. The two
PMTs are modulated at the same frequency as the
laser diode, with the exception of an additional offset
frequency of Dv ¼ 100 Hz. Using the heterodyne
technique, the mixed signals are created to contain
the sum and difference between the signal at the laser
modulation frequency and at 100 Hz higher. Then,
DOI 10.1002/jps
4775
the resulting mixed signals from the PMTs are passed
through two transimpedance amplifiers to filter the
high frequency components, leaving the 100 Hz
signals intact. The remaining phase-shift and ampli-
tude attenuation are then measured. Finally, data
acquisition software is used to acquire the hetero-
dyned signals and record the phase shift (PS),
amplitude (AC), and mean intensity (DC) of the
signal from the sample PMT relative to the reference
PMT. The optical properties of the sampling medium
can then be accurately extracted by solving the
equations for PS, AC, and DC as functions of ma and m0s
at the wavelength of the monochromatic laser.
For the above instrument, Sevick-Muraca and
coworkers18 developed two general methods to
determine the optical properties, including multiple
frequency and multiple distance methods; each
method will be discussed in detail in the following
section. Under different experimental conditions,
individual qualification criteria for each method were
developed to assess the accuracy and precision of
FDPM measurements,18 including (1) whether abnor-
mal measurement error exists during the FDPM
experiment; (2) which ranges of modulation fre-
quency and relative distance are suitable for FDPM
experimentation for a given sample; and (3) which
segments of the measurement can be used to generate
accurate and reliable optical properties.
Mathematical Approaches to Determine la and ls0
Fishkin and Gratton solved the diffusion approxima-
tion to the RTE (Eq. 5) for an infinite and macro-
scopically uniform medium. The outcome was
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
4776 SHI AND ANDERSON
Figure 6. Time evolution of the intensity from a sinu-
soidally intensity-modulated source. The figure was
adapted from Fishkin and Gratton.42
expressions for three experimentally determined
quantities: (1) the steady-state photon density or
the time invariant average intensity, the DC compo-
nent, (2) the amplitude of the photon-density oscilla-
tion, the AC component, and (3) the phase shift of the
photon-density wave, the PS component.42,43 These
three quantities are illustrated in Figure 6.42 To
eliminate measurement error at a given v, the
properties of the photon density wave at two different
source-detector separations, namely, r and r0, are
normally measured and compared.43 Thus, the DC,
AC, and PS are normally measured in their relative
quantities, which are expressed as a function of the
optical coefficients as
surements: the multiple frequency and multiple
distance methods.
Multiple Frequency (MF) Method. Eqs. (15)–(18)
show that at fixed distances between the source and
detector, r and r0, the values of ln[(r/r0)ACrel], PSrel,
and ln(Modrel) are nonlinear functions of the modula-
tion frequency. Thus, measurements of DC, AC, and
PS (and therefore Mod), which are functions of the
modulation frequency at two fixed source-detector
distances, can be used to estimate the optical
properties via nonlinear regression.
Since there are only two unknowns, multiple
combinations of DC, AC, PS, and Mod can be used
to determine ma and m0s . It was found that a regression
approach based on only one type of measurement data
was insufficient to obtain accurate results.18,44 It was
also determined that simultaneous regression of
DC þ PS, AC þ DC þ PS gave comparable results to
simultaneous regression of AC þ PS, while simulta-
neous regression of DC þ AC or Mod þ PS failed to
accurately estimate the optical properties.18,43
Error associated with the nonlinear regression was
investigated via a Monte Carlo method of error
analysis.18 The study used a polystyrene colloidal
suspension to determine m0s and compared it with the
value calculated by Mie theory. Based on the error
analysis, the accuracy and precision of the deter-
mined reduced scattering coefficients obtained via the
multiple frequency method were studied and com-
r
 pared at multiple source-detector distances. The
0 3m ðm þ m0s Þ 1=2 comparison concluded that a minimum relative
ln DCrel ¼ ðr  r0 Þ a a (15)
r 2 distance (r  r0) of about 2.5 mm was necessary for
reasonable accuracy and precision. If the relative
r
 
0 3ma ðma þ m0s Þ 1=2 distance is smaller than this minimum distance
ln ACrel ¼ ðr  r0 Þ threshold, unsatisfactory accuracy and precision of
r 2
2sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 31=2 the extracted m0s will be induced due to the large
 
4 v 2 uncertainty in the FDPM measurement.
 1þ þ 15 ð16Þ
yma
Multiple Distance (MD) Method. Eqs. (15)–(18)
also show that at a fixed modulation frequency, v, the
PSrel ¼ ðr  r0 Þ values of ln[(r/r0)DCrel], ln[(r/r0)ACrel], PSrel, and
2sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 31=2 ln(Modrel) are linear functions of the relative distance
 1=2  2 between the detectors (r  r0). Thus, the slopes (k) can
3ma ðma þ m0s Þ 4 1þ v
  15 be determined from plots of ln[(r/r0)DCrel], ln[(r/
2 yma
r0)ACrel], PSrel, and ln(Modrel) versus (r  r0). Subse-
(17) quently, simultaneous regression via different com-
binations of kDC, kAC, kMod, and kPS can be used to
  obtain the optical properties of the sample medium.
3ma ðma þ m0s Þ 1=2
lnðModrel Þ ¼ ðr  r0 Þ Comparisons were performed between multiple
2
2 0sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 11=2 3 combinations of kDC, kAC, kMod, and kPS to determine
 2 the reduced scattering coefficients via the MD method
6 p ffiffiffi v 7
4 2@ 1þ þ 1A 5 ð18Þ and those calculated by Mie theory.18 Results
yma
indicated that the reduced scattering coefficients
derived from simultaneous regression of DC þ PS,
Eqs. (15)–(18) express two general approaches for AC þ PS, and AC þ DC þ PS agreed well with the
extracting the optical coefficients from FDPM mea- theoretical calculations, but simultaneous fitting of

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010 DOI 10.1002/jps
 SEPARATION OF ABSORPTION AND SCATTERING IN NIRS
DC þ AC or Mod þ PS failed to accurately estimate
the reduced scattering coefficients. The results
obtained via MD linear regression were similar to
those obtained via MF nonlinear regression. This
suggested that PS data combined with DC and/or AC
data provide the most accurate information of the
optical properties. The modulation data (i.e., AC/DC),
however, are not suitable for deriving the optical
parameters, even when they are combined with PS
data.
The uncertainties of determination via the MD
method were derived from error analysis using
Eqs. (15)–(18).45,46 Error analysis was performed to
calculate the precision and accuracy associated with
the of determination of the reduced scattering
coefficient for a polystyrene colloidal suspension
measured at multiple frequencies via the MD
method. Both precision and accuracy indicated that
a modulation frequency greater than 60 MHz was
necessary to obtain reasonable results.18 If the
modulation frequency is less than the threshold,
unsatisfactory accuracy and precision will be induced
as a result of the uncertainty associated with the
FDPM measurement.
In general, the MF and MD methods perform
similarly. The most obvious advantage of the MD
method is that the analytical solution for the optical
parameters can be directly derived without nonlinear
regression. Although a study showed that the
accuracy of the MD method was better than that of
the MF method, the precision was worse.18 The
authors suggested combining the two methods to
increase the signal-to-noise ratio and improve
the accuracy and precision for the estimation of the
optical properties. For a MF measurement, the
experiment can be performed at several different
relative distances (called the combined MF method),
while for a MD measurement, the experiment can be
performed at several different modulation frequen-
cies (called the combined MD method). The combined
approaches were found to improve the accuracy and
precision for the estimation of the optical coefficients.
Related Pharmaceutical Applications
To date, FDPM has been predominately utilized to
separate absorption and scattering in NIR spectral
responses of pharmaceutical samples. A number of
studies were performed by Dr. Sevick-Muraca and
collaborators, where they applied FDPM for the
analysis of particle size in suspensions and powder
media, and for the determination of constituent
concentration in powder mixtures.9–12,16,19–21,47
For particle size analysis, an inversion algorithm
was developed to determine the particle size dis-
tribution (PSD) and volume fraction for noninteract-
ing colloidal suspension samples (<2% volume
DOI 10.1002/jps
4777
fraction) from FDPM measurements.19 When particle
interaction was accounted for by the full polydisperse
hard sphere Percus–Yevick (HSPY) model, FDPM
was capable of determining PSD and volume fraction
for polydisperse interacting suspensions with volume
fractions up to 40%.20,47 The samples used in these
studies were simple laboratory systems such as
polydisperse polystyrene suspensions and titanium
dioxide suspensions. Given these findings, the
potential for on-line analyses of PSD and volume
fraction of concentrated samples within dense process
streams using FDPM is promising.
FDPM was also reported to be a useful technique
for determining the mean particle size of pharma-
ceutical powders.17 The MD method was used to
determine the optical coefficients for lactose powders
with mean particle sizes of 650, 785, and 828 nm. It
was found that the reduced scattering coefficient was
linearly related to the reciprocal of the mean particle
size determined by different reference methods,
including sieve analysis, laser diffraction, and image
analysis. The absorption coefficients were insensitive
to differences in particle size; however, they were
wavelength-dependent. As a result of the linear
relationship between the reduced scattering coeffi-
cient and the reciprocal of the mean particle size, it
was proposed that FDPM can be used for particle
sizing analysis without spectral pretreatment and
chemometric calibration (traditionally used in NIRS).
Because of its noninvasive nature of measurement
without sample preparation, FDPM is a potential tool
for on-line applications.
Multiple studies have reported a linear relation-
ship between the absorption coefficients determined
by FDPM and the chemical concentrations.16,21 Even
when API was mixed with different particle-sized
excipients, the separated absorption and scattering
properties by FDPM captured the linear relationship
between absorption coefficient and API concentra-
tion without effect of particle size and without
further data pretreatment.21 Comparatively, inten-
sity attenuation-based measurement (e.g., NIRS) was
unable to effectively differentiate between light
absorption and scattering processes, which require
spectral pretreatment to enhance the linearity
between spectral intensity and API concentration.
Moreover, due to the minimal noise associated with
measurements in the frequency domain,18,48 the
absorption coefficient determined by FDPM was
sensitive to the API signal from both the low (<1%,
w/w) and ultralow dose (<0.1%, w/w) formula-
tions.16,21 Meanwhile, the reduced scattering coeffi-
cient showed little sensitivity to changes in API
concentration within the same concentration range.
The major limitation of FDPM for monitoring
chemical composition is that the monochromatic laser
source is not sufficient for determining the concen-
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
4778 SHI AND ANDERSON
tration of both excipients and API, especially for
multi-component pharmaceutical matrices. Thus,
multiple wavelengths measurements are necessary
if FDPM is to be widely used for industrial applica-
tions.
Given that FDPM can determine both absorption
and scattering properties (shown by the above
studies), Sevick-Muraca and coworkers16,17,21 pro-
posed it as a tool for on-line monitoring of powder
blending, including both the variation of API
concentrations from changes in the absorption
coefficient, and variation in the packing arrangement
of the powder bed from changes in the reduced
scattering coefficient.
In order to apply a noninvasive measurement
(e.g., FDPM) for monitoring powder blending, one of
the key questions to be answered is the size of
sampling volume. The determination of sampling
volume provides a means to directly compare FDPM
with other analytical measurements, for example,
NIRS and HPLC. Mathematical expressions pre-
dicting the sampling volume of FDPM were devel-
oped for infinite and semi-infinite powder beds via
probability distribution analysis to describe the
propagation of multiply scattered light between a
point source and point detector separated by a
known distance.10 The predicted volume of inter-
rogation was in agreement with that determined by
empirical measurements of FDPM. Based on the
derived equation, the sampling volume of FDPM is
determined by the (1) separation distance between
the incident point source and the point detector; (2)
optical properties of the sample, and (3) modulation
frequency.
The first article using FDPM to monitor powder
blending was published in 2004 by Pan et al.9 In this
article, FDPM was compared to HPLC as an off-line
method to trace the concentration variation of API in
a terazosin powder blend (0.72%, w/w). Thieved
samples were used for both FDPM and HPLC
measurements. Although the off-line sampling pro-
tocol used to monitor powder blending was not ideal,
the paper did present evidence demonstrating the
relationship between sampling volume and blending
variance. Based on the mathematical expression
developed earlier,10 the sampling volume by FDPM
was estimated to be 1.4 cm3, which was shown to be
larger than that determined by both HPLC (0.65 cm3)
and the reported value for optic fiber-based NIR
spectroscopy (<0.3 cm3). A complete-random-mixture
(CRM) model was used to calculate the blending
variance in terms of relative standard deviation. After
using FDPM, HPLC, and NIR to analyze the
homogenous powder mixture, the relative standard
deviations were found to be 4.8%, 7%, and 10%,
respectively. Thus, the expected inverse relationship
between blending variance and sampling volume was
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
clearly illustrated. The larger sampling volume of
FDPM was attributed to the separation between the
source and detector, which is larger when compared
to the close proximity of source and detector
commonly used in optic fiber-based NIRS. Addition-
ally, the instrument error for FDPM was also
determined using repetitive measurements of one
location of a stationary powder bed. The instrument
error was determined to be about 0.12%. When
compared to a blending variance of 4.8%, instrument
noise can be neglected in the calculation of variance.
Given the large sampling volume and low instrument
error, Pan et al. expected FDPM to provide high-
precision determination of low dose API content in
pharmaceutical processing streams.
The most recent pharmaceutical application of
FDPM combined a two-speed diffusion model with
FDPM to allow both absorption and scattering
information to contribute to monitoring concentra-
tion variation during a powder blending process.11,12
It is well known that the solid volume fraction
changes irregularly over time during powder blend-
ing. Monte Carlo simulation and FDPM measure-
ment were used to demonstrate that both the
absorption and reduced scattering coefficients of
the powder beds increased with the increment of
solid-volume fraction.12 Since both the variations of
API concentration and the solid-volume fraction in
the powder blending process contribute to changes in
the absorption coefficient of the powder bed, it may
not be possible to simultaneously determine the
variation of API concentrations and the variation of
the solid-volume fraction when only the absorption
coefficient of the powder bed is considered. This
indicates that, besides the absorption coefficient,
additional information is required to accurately
determining the blend homogeneity. Thus, instead
of using the absorption coefficient, the ratio of the
absorption to the reduced scattering coefficient was
proposed and confirmed to be insensitive to the solid
volume fraction, while still maintaining sensitivity to
API concentration.12 Pan et al. concluded that the
ratio would be appropriate for monitoring powder
blending homogeneity, regardless of the variation of
solid volume fraction.
Spatially-Resolved Spectroscopy (SRS)
Spatially resolved spectroscopy involves the mea-
surement of the spatial (either radial or depth)
distribution of reflectance and transmittance of a
turbid medium to determine its optical properties.
SRS was first used to determine the effective
extinction coefficient ðm0t Þ. This was accomplished
by measuring the fluence-depth distribution in
tissue with broad beam illumination using three
interstitially placed optical fibers connected to
photodetectors.49
DOI 10.1002/jps
 SEPARATION OF ABSORPTION AND SCATTERING IN NIRS
Figure 7. Optical set-up of SRS. The figure was adapted
from Wilson.34
Instrumentation
The most common instrument set-up measures
diffuse reflectance (R) as a function of the radial
distance (r) on the sample surface (i.e., radially
diffused reflectance). The initial set-up used in the
field of tissue optics relied on optic-fibers, in which
point or pencil-beam source was used to perpendicu-
larly illuminate sample surface, and diffusively
reflected signals were detected by optical-fibers at a
specified distances from the illumination spot
(Fig. 7).34 Detection could be accomplished using
one single fiber moving radially and measuring
reflectance one radial distance at a time, or using a
linear fiber array picking up signal simultaneously
from different radial distances. With the development
of imaging technology, charge-coupled device (CCD)
cameras have emerged as a means of detecting
radially diffused reflectance.5,50–52 The advantage
of using CCD cameras to capture the entire radial
distribution of diffusively reflected signals is to
enhance the signal-to-noise ratio of the reflectance
measurements via spatial image processing (i.e.,
signal binning) of equivalent radial distances.50
Mathematical Approaches to Determine la and ls0
In the field of tissue optics, a closed form analytical
equation was developed by Farrell and Patterson53 to
correlate the radially diffused reflectance with ma and
m0s . However, the derived equation was based on the
assumptions and boundary conditions for a semi-
infinite medium, which is not necessarily applicable
for pharmaceutical samples. A semi-infinite homo-
genous medium has optical boundaries that are
infinitely wide, which indicates that the boundaries
are much wider than the spatial extent of the photon
distribution. Therefore, an alternative approach is
needed before SRS can be applied for pharmaceutical
applications.
Numerical methods have also been used to deter-
mine ma and m0s from SRS measurements. For the case
DOI 10.1002/jps
4779
where there is no derived analytical equation
matching the experimental conditions, numerical
simulation (i.e., Monte Carlo simulation-based
photon migration) can be used as an alternative to
estimate the optical coefficients. These techniques
typically involve forward calculation of R(r) over a
expected range of ma and m0s values, followed by either
iterative interpolation of the measured R(r) inside the
calculated range to find the optical properties that
yield the smallest interpolation error,52 or prediction
of the optical properties by certain regression
algorithms, such as artificial neural network
(ANN)54 and partial least square (PLS).5,51
Related Pharmaceutical Applications
Shi and Anderson were the first to explore the
potential applications of SRS in the pharmaceutical
field. They, along with other researchers, published
a series of reports that focused on SRS method
development for pharmaceutical samples,5 the
enhanced understanding that separated optical
coefficients offer to practical uses of NIRS6,7 and
the application of optical coefficients to spectroscopic
analyses under practical conditions.5,8
The authors established a chemical imaging-based
spatially resolved spectroscopic measurement.5 A
chemical imaging system was used to capture both
the spatial and spectral information from the radially
diffused reflectance of pharmaceutical solid samples
(either as powder or tablets). Subsequently, a Monte
Carlo simulation-oriented PLS model was used to
predict the optical coefficients from the measured
radially diffused reflectance. Simulation and reference
correction by Intralipid at 1064 nm normalized the
simulated radially diffused reflectance such that it was
comparable to the measured counterpart. This com-
parability indicated that the model based on simulated
data could be applied to the radially diffused reflec-
tance measurements to predict the optical coefficients.
The optical coefficients extracted from SRS have
been used to enhance the understanding of practical
applications of NIRS.6 The samples used here were
pharmaceutical powders of various particle sizes or
compacts of various densities. An increase in either
particle size or tablet density induced a proportional
change in ma and an inversely proportional change in
m0s . The separated ma and m0s were input into the
Monte Carlo simulation-based photon migration
program to trace the photon absorption behavior
and record the depth of penetration. The consistency
observed between the measured and simulated
results indicated that the ma and m0s were the
dominant factors in the NIR absorbance profile and
the depth of penetration characteristics, respectively.
The combination of optical coefficients determined
by SRS and Monte Carlo simulation-based photon
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
4780 SHI AND ANDERSON
migration provides a unique tool to understand the
depth- and radially resolved profiles of NIR radiation
on pharmaceutical samples.7 The depth-/radially
resolved profile exploits the relationship between
the cumulative percentage of reflected information
and the depth/radial distance in a sample matrix.
In silico studies revealed that both the depth- and
radially resolved profiles are nonlinear, indicating
that portions of the sample close to the point of
interest, along either the depth/the radial distance,
contribute more to the final reflectance than those
further away. The nonlinearity of the profiles is
expected to be dependent on the ma and m0s at their
corresponding wavelength. Additionally, the simu-
lated depth-/radially resolved profile was also applic-
able to chemical imaging systems. The depth and
radial distance corresponding to 95% of the reflected
information were determined to be approximately 150
and 300 mm, respectively, for the simulation condi-
tions. These values were larger than the physical size
of a single pixel in any commercially available
chemical imaging system. Thus, the observed infor-
mation from a single pixel was believed to be
representative of the information within a specific
3-D volume. In other words, the reflected intensity
captured in a given pixel of a chemical image is a
weighted average across a specific depth and radial
distance. These results underscore the precautions
that must be taken when interpreting NIR chemical
images.
Based on the enhanced understanding by the above
studies about individual roles of absorption and
scattering in NIRS, the determined ma and m0s were
subsequently applied to spectroscopic analyses under
practical conditions. Due to the wavelength and
absorption dependency of m0s , a m0s -based scattering
correction method was proposed5 as an alternative to
wavelength-independent scattering correction meth-
ods, such as SNV and MSC. When applied to model the
chemical compositions of tablets, the m0s -based scatter-
ing correction method, termed scattering orthogona-
lization, resulted in superior calibration and prediction
statistics compared to SNV. The enhanced perfor-
mance of scattering orthogonalization was attributed
to its ability to mitigate the physical interferences
while preserving the chemical information. Therefore,
this method is expected to be useful for routine model
calibration and model update procedures as it mini-
mizes changes to the calibration resulting from
physical variations in the samples related to the m0s .
Since pure component materials are typically
available in the pharmaceutical industry, both ma
and m0s of a pure component raw material can be used
to represent interfering signals when predicting the
chemical concentrations of other components within a
powder or tablet mixture. In a recent paper, ma and m0s
were integrated into specific chemometric algo-
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
rithms.8 For example, net analyte signal (NAS) and
generalized least squares (GLS) were used to simplify
a NIRS multivariate calibration model using only
pure component spectra and concentration values
from one formulation mixture. It was found that the
simplified model was conducive to parsimonious
multivariate models and reached the same or even
lower prediction error than traditional approaches.
Thus, optical coefficient-based signal processing is
expected to be beneficial to both calibration and
update efforts during routine NIR spectroscopic
analyses.
COMPARISON AMONG TECHNIQUES USED
TO SEPARATE ABSORPTION AND SCATTERING
IN NIRS
Five categories of techniques have been applied to
pharmaceutical samples to separate absorption and
scattering in NIRS. To simplify the following discus-
sion, these techniques can be reorganized into two
major groups: time and intensity related measure-
ments. TRS and FDPM are both time dependent
measurements, while integrating sphere-based
reflectance and transmittance measurements, mea-
surements through layers of material of different
thicknesses and SRS are intensity based techniques.
The relationship between TRS and FDPM can be
described as follows.48 A broadened pulse will be
observed in the time domain h(t), if an infinitesimally
short pulse is applied to a turbid scattering medium.
Alternatively, if a sinusoidally modulated light source
is applied to the same medium, the photon flux at the
detector will also be sinusoidal in time, but the
oscillation will be delayed in phase and amplitude
relative to the source. In essence, the time domain
signal h(t) can be linked to the phase and amplitude by
the Fourier transform such that any information
acquired in the time domain can also be, in principle,
obtained in the frequency domain. However, certain
practical differences between these two techniques do
exist.48 First, typical FDPM measurements using
frequencies of 300 MHz or less are considerably less
expensive than the time-resolved techniques. Second,
phase and amplitude measurements can be made in
near-real time such that the influence of time-varying
phenomena can be studied in a sample medium.
Comparatively, the acquisition rate of time-resolved
data collected using a time-correlated single photon
counter is usually limited by electronic constraints
imposed by the count rate. Therefore, while the funda-
mental observations are the same, the underlying
details of the two techniques dictate their applicability.
The integrating sphere-based approach, measure-
ment through layers of material of different
thicknesses and SRS are all intensity based measure-
DOI 10.1002/jps
 SEPARATION OF ABSORPTION AND SCATTERING IN NIRS
ments. The first two techniques detect reflected signals
exiting the sample surface and record these signals as a
single magnitude at a given wavelength. This method
is referred to as the total diffuse reflectance measure-
ment.34 SRS, on the other hand, measures radially-
diffused reflectance, which is the individual reflec-
tance signal at a specific radial distance for a given
wavelength. This method is referred to as the local
diffuse reflectance measurement.34
Limited studies have been reported to date to
compare the practical performance of different
techniques across the same sample platform (i.e.,
the same analyte of interest). Swartling et al.55
compared the practical performance of the integrat-
ing sphere-based approach, TRS and SRS for
determining the optical coefficients of a set of tissue
phantom samples. Their study was limited to a
wavelength range of 660–785 nm. The integrating
sphere-based method was shown to be the best
approach to estimate the reduced scattering coeffi-
cients. The authors’ results were supported by
previously reported values for phantom samples.56
The integrating sphere-based method, however, had a
poor limit of detection for determining absorption
coefficients. Comparatively, TRS demonstrated the
capacity to determine low absorption coefficients.
Overall, Swartling et al. concluded that the differ-
ences between the approaches for the determination
of the optical coefficients were minimal.
Sevick-Muraca et al.57 investigated the theoretical
differences between the time dependent measure-
ments (TRS or FDPM) and SRS. The authors
concluded that SRS, unlike TRS and FDPM, does
not provide direct measurements of photon path
length, and it relies solely on the detection of light
intensity attenuation to describe the absorption and
scattering behaviors. Compared to SRS, TRS, and
FDPM do not measure relative intensity, but rather
absolute ‘‘time-of-flight’’ and phase delay. Thus, TRS
and FDPM are essentially self-calibrating, and are
not subjected to the measurement errors associated
4781
Second, the accuracy of the determined optical
coefficients is dependent upon the mathematical
approaches used. For instance, nonlinear regression
performed on multiple, rather than two, radial
distance points may enhance the robustness of SRS
for extracting the optical coefficients.55
CONCLUSIONS AND PERSPECTIVES
Overall, publications detailing the separation of
absorption and scattering phenomena in NIRS have
improved the current understanding of NIR diffuse
reflectance, especially with regard to the individual
roles of absorption and scattering. The enhanced
understanding has provided and continuously will
offer the basis for improved spectroscopic analyses
under practical conditions.
With the increasing awareness of the importance of
NIRS in PAT, the spectroscopic application with
mechanistic understanding of underlying optical
phenomena is expected to save time and effort when
integrating PAT into pharmaceutical processes,
and enhance the robustness of multivariate models
to provide effective process monitoring and control
to ultimately improve end-product quality. For
instance, a spectral library of ma(l) and m0s ðlÞ of pure
component materials is expected to provide tremen-
dous leverage for both multivariate calibration and
routine calibration update in pharmaceutical appli-
cations of NIRS. Additionally, upcoming generations
of NIRS instrumentation are expected to integrate
the techniques used in the separation of absorption
and scattering, such as those reviewed in this article,
to delineate NIR absorbance spectra directly into
absorption and scattering profiles, which will simplify
subsequent qualitative and quantitative applications.
In the meantime, improvements to the techniques
used for the separation of absorption and scattering in
NIRS are necessary.
with the calibration with respect to an external 
standard.
In summary, there are two main reasons for the
limited number of studies comparing the practical
performances of the various techniques for separating
absorption and scattering. First, optical set-ups
interrogate different sample volumes.55 For instance,
because of the large source-detector distance, FDPM
may interrogate a larger sample volume compared to
the other techniques. A larger sampling volume
minimizes the potential effects of sample heteroge-
neity on the resultant optical coefficients, leading to
better precision and reduced measurement error. The
effects of inhomogeneity are also mitigated when
A standard with known ma(l) and m0s ðlÞ in NIR
range should be developed to improve the accu-
racy of individual measurements and provide a
platform to compare measurements across dif-
ferent techniques. The most common standard in
the field of tissue optics is Intralipid. However,
the wavelength range used in tissue optics is
narrow (600–1100 nm) compared to that which
is typically used in pharmaceutical applications
(780–2500 nm). Therefore, the determination of
ma and m0s for Intralipid over the NIR spectral
range, or the design and measurement of some
new standard, will be essential for continuous
improvement of the techniques reviewed herein.
transmittance rather than reflectance is used in TRS  Many of the individual techniques might also
as the former often interrogates larger volumes.23 benefit from advances in instrumentation.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
4782 SHI AND ANDERSON
For example, the extended wavelength range
covered by TRS or instrumentation offering
simultaneous FDPM measurements over multi-
ple wavelengths, or better yet, a continuous
wavelength range, is expected to improve the
functionally and generality of these techniques
in pharmaceutical applications.
Finally, the idea of applying the separation of
absorption and scattering in NIRS to enhance the
mechanistic understanding of the fundamental opti-
cal phenomena and improve the practical spectro-
scopic analyses is expected to facilitate future
applications of NIRS.
ACKNOWLEDGMENTS
The authors would like to acknowledge Dr. Steve
Short for his skillful scientific and grammatical edit-
ing, which has been helpful in preparing this manu-
script.
REFERENCES
1. Reich G. 2005. Near-infrared spectroscopy and imaging: Basic
principles and pharmaceutical applications. Adv Drug Deliv
Rev 57:1109–1143.
2. Cogdill RP, Drennen JK. 2006. Near-Infrared Spectroscopy. In:
Brittain H, editor. Spectroscopy of pharmaceutical solids. New
York: Taylor & Francis. pp. 313–412.
3. Mobley J, Vo-Dinh T. 2003. Optical properties of tissue. In:
Vo-Dinh T, editor. Biomedical photonics handbook. Boca Raton:
CRC Press. pp. 2.1–2.75.
4. Welch AJ, van Gemert MJC, Star WM, Wilson BC. 1995.
Definitions and overview of tissue optics. In: Welch AJ, van
Gemert MJC, editors. Optical-thermal response of laser-irra-
diated tissue. New York: Plenum Press. pp. 15–46.
5. Shi ZQ, Anderson CA. 2009. Scattering orthogonalization of
near-infrared spectra for analysis of pharmaceutical tablets.
Anal Chem 81:1389–1396.
6. Shi Z, Anderson CA. 2009. Application of Monte Carlo simula-
tion-based photon migration for enhanced understanding of
near-infrared (NIR) diffuse reflectance. Part I: Information
depth in pharmaceutical materials. J Pharm Sci 99:2399–2412.
7. Shi Z, Anderson CA. 2009. Application of Monte Carlo simula-
tion-based photon migration for enhanced understanding of
near-infrared (NIR) diffuse reflectance. Part II: Photon radial
diffusion in NIR chemical images. J Pharm Sci 10.1002/
jps.22125.
8. Shi Z, Cogdill RP, Martens H, Anderson CA. 2009. Optical
coefficient-based multivariate calibration on near-infrared
spectroscopy. J Chemometrics 24:288–299.
9. Pan T, Barber D, Coffin-Beach D, Sun Z, Sevick-Muraca EM.
2004. Measurement of low-dose active pharmaceutical ingre-
dient in a pharmaceutical blend using frequency-domain
photon migration. J Pharm Sci 93:635–645.
10. Pan T, Sevick-Muraca EM. 2002. Volume of pharmaceutical
powders probed by frequency-domain photon migration mea-
surements of multiply scattered light. Anal Chem 74:4228–
4234.
11. Pan TS, Dali SS, Sevick-Muraca EM. 2005. Evaluation of
photon migration using a two speed model for characterization
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010
of packed powder beds and dense particulate suspensions. Opt
Express 13:3600–3618.
12. Pan TS, Sevick-Muraca EM. 2006. Evaluation of ingredient
concentration in powders using two-speed photon migration
theory and measurements. J Pharm Sci 95:530–541.
13. Sevick-Muraca EM, Sun ZG, Huang YQ. 2005. Method for
characterizing particles in suspension from frequency domain
photon migration measurements. US Patent 6,930,777.
14. Sevick-Muraca EM, Sun ZG, Huang YQ. 2007. Method for
characterising particles in suspension from frequency domain
photon migration measurements. US Patent 7,268,873.
15. Sevick-Muraca EM, Sun ZG, Pan TS. 2004. Characterizing
powders using frequency-domain photon migration. US Patent
6,771,370.
16. Shinde RR, Balgi GV, Nail SL, Sevick-Muraca EM. 1999.
Frequency-domain photon migration measurements for quan-
titative assessment of powder absorbance: A novel sensor of
blend homogeneity. J Pharm Sci 88:959–966.
17. Sun Z, Torrance S, McNeil-Watson FK, Sevick-Muraca EM.
2003. Application of frequency domain photon migration to
particle size analysis and monitoring of pharmaceutical pow-
ders. Anal Chem 75:1720–1725.
18. Sun ZG, Huang YQ, Sevick-Muraca EM. 2002. Precise analysis
of frequency domain photon migration measurement for char-
acterization of concentrated colloidal suspensions. Rev Sci
Instrum 73:383–393.
19. Sun ZG, Sevick-Muraca EM. 2001. Inversion algorithms for
particle sizing with photon migration measurement. AIChE J
47:1487–1498.
20. Sun ZG, Tomlin CD, Sevick-Muraca EM. 2001. Approach for
particle sizing in dense polydisperse colloidal suspension using
multiple scattered light. Langmuir 17:6142–6147.
21. Torrance SE, Sun Z, Sevick-Muraca EM. 2004. Impact of
excipient particle size on measurement of active pharmaceu-
tical ingredient absorbance in mixtures using frequency
domain photon migration. J Pharm Sci 93:1879–1889.
22. Abrahamsson C, Lowgren A, Stromdahl B, Svesson T, Anders-
son-Engels S, Johansson J, Folestad S. 2005. Scatter correction
of transmission near-infrared spectra by photon migration
data: Quantitative analysis of solids. Appl Spectrosc 59:
1381–1387.
23. Johansson J, Folestad S, Josefson M, Sparen A, Abrahamsson
C, Andersson-Engels S, Svanberg S. 2002. Time-resolved NIR/
Vis spectroscopy for analysis for solids: Pharmaceutical tablets.
Appl Spectrosc 56:725–731.
24. Abrahamsson C, Svensson T, Svanberg S, Andersson-Engelsen
S, Johansson J, Folestad S. 2004. Time and wavelength
resolved spectroscopy of turbid media using light continuum
generated in a crystal fiber. Opt Express 12:4103–4112.
25. Chauchard F, Roger JM, Bellon-Maurel V, Abrahamsson C,
Andersson-Engels S, Svanberg S. 2005. MADSTRESS: A linear
approach for evaluating scattering and absorption coefficients
of samples measured using time-resolved spectroscopy in
reflection. Appl Spectrosc 59:53–59.
26. Chauchard F, Roussel S, Roger JM, Bellon-Maurel V, Abra-
hamsson C, Svensson T, Andersson-Engels S, Svanberg S.
2005. Least-squares support vector machines modelization
for time-resolved spectroscopy. Appl Opt 44:1–7.
27. Abrahamsson C, Johansson J, Andersson-Engels S, Svanberg
S, Folestad S. 2005. Time-resolved NIR spectroscopy for quan-
titative analysis of intact pharmaceutical tablets. Anal Chem
77:1055–1059.
28. Burger T, Ploss HJ, Ebel S, Fricke J. 1997. Diffuse reflectance
and transmittance spectroscopy for the quantitative determi-
nation of scattering and absorption coefficients in quantitative
powder analysis. Appl Spectrosc 51:1323–1329.
29. Burger T, Kuhn J, Caps RJF. 1997. Quantitative determina-
tion of the scattering and absorption coefficients from diffuse
DOI 10.1002/jps
 SEPARATION OF ABSORPTION AND SCATTERING IN NIRS
reflectance and transmittance measurements: Application to
pharmaceutical powders. Appl Spectrosc 51:309–317.
30. Kuhn J, Korder S, Arduini-Schuster MC, Caps R, Fricke J.
1993. Infrared-optical transmission and reflection measure-
ments on loose powders. Rev Sci Instrum 64:2523–2530.
31. Burger T, Fricke J, Kuhn J. 1998. NIR radiative transfer
investigations to characterise pharmaceutical powders and
their mixtures. J Near Infrared Spectrosc 6:33–40.
32. Dahm DJ, Dahm KD. 1999. Spectrographic analysis instru-
ment and method based on discontinuum theory United States
Patent 5,912,730.
33. Kessler W, Oelkrug D, Kessler RW. 2008. Using scattering and
absorption spectra as MCR-hardmodel constraints for diffuse
reflectance measurements of tablets. 11th International Con-
ference on Chemometrics for Analytical Chemistry, Montpel-
lier, France.
34. Wilson BC. 1995. Measurement of tissue optical properties:
methods and theories. In: Welch AJ, van Gemert MJC, editors.
Optical-thermal response of laser-irradiated tissue. New York:
Plenum Press. pp. 233–274.
35. Abrahamsson C. 2005. Dissertation: Time-resolved spectro-
scopy for pharmaceutical applications. Department of Physics.
Lund: Lund University. pp. 29–36.
36. Nichols MG. 1996. Dissertation: Transport of oxygen and light
in model tumor systems. Department of Physics and Astron-
omy. Rochester: University of Rochester. pp. 84–98.
37. Nichols MG, Hull EL, Foster TH. 1997. Design and testing of a
white-light, steady-state diffuse reflectance spectrometer for
determination of optical properties of highly scattering sys-
tems. Appl Opt 36:93–104.
38. Wang LH, Jacques SL, Zheng LQ. 1995. MCML—Monte Carlo
modeling of light transport in multi-layered tissues. Comput
Methods Programs Biomed 47:131–146.
39. Weinman JA, Shipley ST. 1972. Effects of multiple scattering
on laser pulses transmitted through clouds. J Geophys Res
77:7123–7128.
40. Patterson MS, Chance B, Wilson BC. 1989. Time resolved
reflectance and transmittance for the noninvasive measure-
ment of tissue optical properties. Appl Opt 28:2331–2336.
41. van Staveren HJ, Moes CJM, van Marle J, Prahl SA, van
Gemert MJC. 1991. Light scattering in Intralipid-10% in the
wavelength range of 400–1100 nm. Appl Opt 30:4507–4514.
42. Fishkin JB, Gratton E. 1993. Propagation of photon-density
waves in strongly scattering media containing an absorbing
semi-infinite plane bounded by a straight edge. J Opt Soc Am A
10:127–140.
43. Fishkin JB, So PTC, Cerussi AE, Fantini S, Franceschini MA,
Gratton E. 1995. Frequency-domain method for measuring
spectral properties in multiple-scattering media: Methemoglo-
bin absorption spectrum in a tissuelike phantom. Appl Opt
34:1143–1155.
44. Pham TH, Coquoz O, Fishkin JB, Anderson E, Tromberg BJ.
2000. Broad bandwidth frequency domain instrument for quan-
titative tissue optical spectroscopy. Rev Sci Instrum 71:2500–
2513.
DOI 10.1002/jps
4783
45. Fantini S, Franceschini MA, Fishkin JB, Barbieri B, Gratton E.
1994. Quantitative determination of the absorption spectra of
chromophores in strongly scattering media: A light-emitting-
diode based technique. Appl Opt 33:5204–5213.
46. Gerken M, Faris GW. 1999. High-precision frequency-domain
measurements of the optical properties of turbid media. Opt
Express 24:930–932.
47. Dali SS, Rasmussen JC, Huang YQ, Roy R, Sevick-Muraca EM.
2005. Particle sizing in dense suspensions with multiwave-
length photon migration measurements. AIChE J 51:1116–
1124.
48. Patterson MS. 1995. Principles and applications of frequency-
domain measurements of light propagation. In: Welch AJ, van
Gemert MJC, editors. Optical-thermal response of laser-irra-
diated tissue. New York: Plenum Press. pp. 333–364.
49. Doiron DR, Svaasand LO, Profio AE. 1983. Light dosimetry in
tissue: application to photoradiation therapy. In: Kessel D,
Dougherty TJ, editors. Porphyrin photosensitization. New
York: Plenum Press. pp. 63–76.
50. Kienle A, Lilge L, Patterson MS, Hibst R, Steiner R, Wilson BC.
1996. Spatially resolved absolute diffuse reflectance measure-
ments for noninvasive determination of the optical scattering
and absorption coefficients of biological tissue. Appl Opt 35:
2304–2314.
51. Pham TH, Eker C, Durkin A, Tromberg BJ, Andersson-Engels
S. 2001. Quantifying the optical properties and chromophore
concentrations of turbid media by chemometric analysis of
hyperspectral diffuse reflectance data collected using a Fourier
interferometric imaging system. Appl Spectrosc 55:1035–
1045.
52. Pham TH, Bevilacqua F, Spott T, Dam JS, Tromberg BJ,
Andersson-Engels S. 2000. Quantifying the absorption and
reduced scattering coefficients of tissuelike turbid media over
a broad spectral range with noncontact Fourier-transform
hyperspectral imaging. Appl Opt 39:6487–6497.
53. Farrell TJ, Patterson MS. 1992. A diffusion theory model of
spatially resolved, steady-state diffuse reflectance for the non-
invasive determination of tissue optical properties in vivo. Med
Phys 19:879–888.
54. Farrell TJ, Wilson BC, Patterson MS. 1992. The use of a neural
network to determine tissue optical properties from spatially
resolved diffuse reflectance measurements. Phys Med Biol 37:
2281–2286.
55. Swartling J, Dam JS, Andersson-Engels S. 2003. Comparison of
spatially and temporally resolved diffuse-reflectance measure-
ment systems for determination of biomedical optical proper-
ties. Appl Opt 42:4612–4620.
56. Dam JS, Dalgaard T, Fabricius PE, Andersson-Engels S. 2000.
Multiple polynomial regression method for determination of
biomedical optical properties from integrating sphere measure-
ments. Appl Opt 39:1202–1209.
57. Sevick EM, Chance B, Leigh J, Nioka S, Maris M. 1991.
Quantitation of time- and frequency-resolved optical spectra
for the determination of tissue oxygenation. Anal Biochem
195:330–351.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 12, DECEMBER 2010