Review

Analytical Techniques for Quantification of Amorphous/

Crystalline Phases in Pharmaceutical Solids
BIRJU SHAH, VASU KUMAR KAKUMANU, ARVIND K. BANSAL
Department of Pharmaceutical Technology (Formulations), National Institute of Pharmaceutical Education and Research (NIPER),
Sector-67, Phase-X, S.A.S. Nagar, Punjab 160 062, India

Received 30 January 2006; revised 20 March 2006; accepted 27 March 2006

Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20644

ABSTRACT: The existence of different solid-state forms such as polymorphs, solvates,

hydrates, and amorphous form in pharmaceutical drug substances and excipients, along
with their downstream consequences in drug products and biological systems, is well
documented. Out of these solid states, amorphous systems have attracted considerable
attention of formulation scientists for their specific advantages, and their presence,
either by accident or design is known to incorporate distinct properties in the drug
product. Identification of different solid-state forms is crucial to anticipate changes in the
performance of the material upon storage and/or handling. Quantitative analysis of
physical state is imperative from the viewpoint of both the manufacturing and the
regulatory control aimed at assuring safety and efficacy of drug products. Numerous
analytical techniques have been reported for the quantification of amorphous/crystalline
phase, and implicit in all quantitative options are issues of accuracy, precision, and
suitability. These quantitative techniques mainly vary in the properties evaluated, thus
yielding divergent values of crystallinity for a given sample. The present review provides
a compilation of the theoretical and practical aspects of existing techniques, thereby
facilitating the selection of an appropriate technique to accomplish various objectives of
quantification of amorphous systems. 2006 Wiley-Liss, Inc. and the American Pharmacists
Association J Pharm Sci 95:16411665, 2006
Keywords: amorphous; X-ray powder diffractometry; thermal analysis; spectroscopy;
solution calorimetry; crystallinity; calorimetry (ITC); calorimetry (DSC)

INTRODUCTION affords protection to organisms against harsh

Once being an exclusive domain of ceramic and
polymer science,1 glassy state is now extensively
studied in diverse fields like basic physics, electro-
nics, metallurgy, space research, food sciences,2
and pharmaceutical sciences.3,4 Vitrification or
glass formation is the manner in which nature
Correspondence to: Arvind K. Bansal (Telephone: 91-0-172
2214682-87; Fax: 91-0-172 2214692;
E-mail: akbansal@niper.ac.in)
Journal of Pharmaceutical Sciences, Vol. 95, 16411665 (2006)
2006 Wiley-Liss, Inc. and the American Pharmacists Association
climatic conditions,5 the strategy mankind uses to
preserve food stuffs,6 the means by which highly
pure semiconductors and optics are prepared,7
and also the form in which most water is present
in the universe.8
Amorphous or glassy solids,9 also called as
disordered systems10 (due to randomness in molec-
ular conformation) and frustrated systems11 (due
to geometric and symmetry frustration at molec-
ular level)12 are usually defined with reference to a
crystalline solid. An amorphous solid lacks any
long-range translational orientation symmetry
that characterizes crystalline structures13

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006 1641

1642 SHAH, KAKUMANU, AND BANSAL
although it may have short range molecular order
similar to a crystalline solid. Thus amorphous
systems can be thought of as liquids that have
been solidified by the removal of thermal energy
or a solvent, in a manner that circumvents
crystallization.10
Amorphous solids have attracted the interest of
pharmaceutical scientists because of two develop-
ments:13 (i) there has been a continuous increase in
the number of insoluble molecules in the develop-
mental pipeline because of the advent of novel
methods of synthesizing and screening potential
drug molecules;14 and (ii) a growing attention
especially in regulatory aspects of the pharmaceu-
tical solids.15 By virtue of their high energy, the
amorphous systems provide enormous benefits of
solubility and compressibility. There is virtually
no limit to the type of materials, which can be made
amorphous, thus providing an approach to
enhance the absorption and performance of poorly
soluble drugs. Over 24 active pharmaceuticals and
excipients are listed in the current European
pharmacopoeia as being amorphous, and more
than 25 marketed products (including tablets,
capsules, injectables, and powders) are described
as amorphous in Physicians Desk Reference.10
However, the full commercial potential of the
advantages offered by these amorphous systems
has not been realized because of their inherent
physical and chemical instability and lack of
characterization tools.12 Attempts to stabilize
amorphous systems by various approaches have
been handicapped due to lack of analytical techni-
que, capable of discerning subtle changes in lattice
order in substantially amorphous materials.10
The preparation of amorphous nature can be
explained in a three-step procedure, involving (i)
energization of material, (ii) de-energization of
material, and (iii) kinetic trapping of the amor-
phous form.16 Kinetically high-cooling rates,
above a critical value, allow a molten material to
become a frozen liquid or vitrify without crystal-
lization.13 Amorphous solids can be generated
from different states of matter utilizing various
techniques, like particle size reduction, desolva-
tion of crystalline form, precipitation from solu-
tion, solidification of melt, and condensation of
vapor.10,17
The glass transition temperature (Tg) is the
single most defining characteristic of amorphous
systems. It represents the temperature at which
the material is kinetically unable to attain equili-
brium in the time scale of measurement, as it loses
its thermal energy, resulting in a change in the
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
temperature dependence of the enthalpy and
volume. This higher internal energy confers the
amorphous state with enhanced thermodynamic
properties relative to the crystalline state (e.g.,
solubility, vapor pressure, and specific heat) and
greater molecular motion, hence greater chemical
reactivity and a tendency to spontaneously crys-
tallize, possibly at different rates above and
below Tg.18
Thus, despite having a molecular level struc-
ture akin to that of liquids, amorphous materials
have unique macroscopic properties and hence can
provide advantages of both material states.10
Compared to their traditionally used crystalline
counterparts, these disordered systems differ in
solubility, stability, dissolution behavior, hygro-
scopicity, and physicotechnical properties. These
distinctive properties provide attractive avenues
of drug delivery to formulation scientists while
overcoming some of their limitations like inherent
physical instability.12
NEED FOR QUANTIFICATION OF
AMORPHOUS PHASE
The amorphous state can arise in three sets of
circumstances: (i) the drug or excipient may be
deliberately produced in an amorphous form to
enhance product performance. The use of plasti-
cizers, including water to lower the Tg of various
cellulosic and acrylic polymers utilized in film
coating and the use of spray-dried lactose and
microcrystalline cellulose as direct compression
excipients is an apt example of this.18 Other
important examples of this strategy include the
preparation of glassy drugs for enhanced dis-
solution behavior; (ii) the material may be
intrinsically amorphous (at least partially) at
room or body temperature, examples including
various polymers like D:L polylactic acid, poly-
vinylpyrrolidone, or polyethylene glycol; (iii) the
crystalline state maybe inadvertently rendered
amorphous by introduction of varying degrees of
stressmechanical or thermal during various
steps of processing like size reduction,1921
heat drying,22 spray drying,22 granulation,23 and
compression.20,24
The amorphous phase can either occur through-
out the particle, or in parts of it like on the surface.
Accidentally produced amorphous state is predo-
minantly a surface phenomenon, thereby present-
ing a challenging scenario,25 as the disorder may
DOI 10.1002/jps
 QUANTIFICATION OF AMORPHOUS/CRYSTALLINE PHASES
be too small to be easily detected but sufficiently
large to cause significant changes in product
performance26 like postcompression hardness,27
enhanced dissolution rate,28 reduced chemical
stability,21,29,30 and moisture-induced recrystalli-
zation during storage.3133
Consequently, it is important to monitor and
characterize the extent of crystallinity or disorder
during various processing steps like bulk material
scale up, formulation development, manufactur-
ing, and over the intended shelf life of pharma-
ceutical product to ensure a robust and safe
formulations by understanding the behavior of
these amorphous systems.34 Current research in
this area is focused towards identification of
applicability of different techniques for quantify-
ing low-amorphous contents in a sample. Also,
efforts are ongoing to search alternative methods
for determining crystallinity that are accurate,
nondestructive, fast, and require minimal sample
handling. The amorphous state differs from its
crystalline counterpart in many physicochemical
properties such as lattice disorder, higher aqueous
solubility, water sorption capacity, chemical reac-
tivity, and molecular mobility18 and hence, any
such property that varies proportionately with the
fraction of crystalline phase in crystalline/amor-
phous mixtures can be used to measure crystal-
linity.30
QUANTIFICATION TECHNIQUES
Over the years, various analytical techniques
have been reported for quantifying amorphous
or crystalline phase in solids. These techniques
vary in their working principles, mechanical and
thermal stress that is applied to the sample, time
of analysis and amount of sample required,
sensitivity of the technique to minute changes,
and the necessity of internal or external stan-
dards.25 Numerous considerations are involved in
the development of a good analytical method like
sensitivity, specificity, method ruggedness, and
the skill of the analyst.
The quantitative approaches employed in these
techniques include degree of crystallinity, the
disruption index, and the entropy of processing.35
Degree of crystallinity can be defined as the
percent crystalline content of the total sample.36
The disruption index (d.i.) is the rate of change of
the difference between the entropy of the solid and
that of the liquid, with respect to the ideal entropy
of mixing of the components of the solid.37 The
DOI 10.1002/jps
1643
difference between the entropy of the solid sample
under investigation and the entropy of the same
amount of a reference sample (usually a pharma-
copeial reference standard) is designated as the
entropy of processing, DSP or the entropy of
crystal imperfection.38
However, of the three, degree of crystallinity
is the most widely used approach towards the
quantification of disorder in pharmaceutical sys-
tems. The quantification techniques reported in
literature are classified in Table 1. The amorphous
content of a material is inferred from the degree of
crystallinity of that material in most of the
techniques. This is because these techniques
detect the specific properties of the crystalline
material, except few, where predominantly the
amorphous phase is detected.
Powder X-Ray Diffractometry (PXRD)
Diffraction techniques are perhaps the most
definitive method of detecting and quantifying
molecular order in any system, and conventional,
wide-angle and small-angle diffraction techniques
have all been used to study order in systems of
pharmaceutical relevance.22,30,32 Conventional X-
ray powder diffraction, also known as PXRD can
be used to quantify noncrystalline material down
to levels of 5% and with temperature and
environmental control can also be used to follow
the kinetics of phase transformation. Small-angle
X-ray measurements have been used to study
subtle structural (density) changes in polymers in
the glassy state upon annealing, and neutron
scattering is gaining wider use in the character-
ization of short-range two-dimensional order in
amorphous materials. However, it is important to
consider that the diffraction techniques only see
molecular order, and thus the disorder is only
implied by the absence of order.18
PXRD is one of the most widely attempted
quantification techniques because of its simplicity
and it measures differences in periodicities of
atoms/molecules in a powder sample.40 PXRD
patterns of crystalline forms show strong diffrac-
tion peaks, whereas amorphous ones exhibit
diffuse and halo diffraction patterns. Diffraction
is defined as a scattering phenomenon in which the
incident X-rays, depending upon the phase differ-
ence, are reinforced to form diffracted beams.41
According to Braggs law, diffraction will occur
when the conditions of Eq. 1 are satisfied
nl 2d sin y 1
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
1644 SHAH, KAKUMANU, AND BANSAL

Applicable only
where d is the distance between the planes in a

compounds
containing
306000

500700

to carbon
ss-NMR crystal, expressed in angstrom units, n is the

0.5%

Both
Yes
No

No

No
order of reflection (an integer), and l is the
wavelength of X-rays.
X-ray procedures for the estimation of degree of
crystallinity are based upon the measurement of

technique to IR
complementary
quantification
X-ray scattering from the entire sample including

employed as
FT-Raman

the crystalline and amorphous region of the

6120

Both
2000
<1%

sample. According to the procedure developed by

No
No
No

No

Currently
Hermans and Weidinger,41 the experimentally
measured crystalline and amorphous intensities
(Ic and Ia) are proportional to the crystalline and
amorphous fraction of the sample (xc and xa).
subtraction of
Therefore,
overlap due
Quantitative

to matrix
1060

12%

required
550

Both

xc pIc ; and xa qIa 2

No
No
No

No
IR

where xc and xa are the crystalline and amorphous

fractions, respectively, and p and q are propor-
tionality constants. The sum of crystalline and
several processes
quantification of

occurring during

amorphous fractions (x) is:

dissolution

x xc xa
60120

1070

Possibility of thermal Lack of specificity; Difficulty in

Both
Yes
Yes

Yes
1%
SC

No

::: qIa x
pIc

x pIc
::: Ia

conditions and

q q
experimental
Table 1. Comprehensive Presentation of Various Quantification Techniques

methodology
Amorphous

selection of

A plot of measured values of Ia against those of Ic

30240

20300
0.5%
IMC

Yes
Yes

Yes

Careful
No

for samples of varying degrees of crystallinity will

result in a straight line with slope of p/q. Degree of
crystallinity (xcr) is defined as the percent crystal-
line content of the total sample and is given by the
sample during the

following equation:
scan; crystallinity
changes in the

melting point

xc  100 Ic  100
measured at

xcr 3
640

410

Both
5%
DSC

x Ic qIa =p
Yes
Yes
No

No

The degree of crystallinity can also be determined

from the following approximate expression
Ic  100
Xcr 4
affected by the
dimensions of

Ic Ia
Crystalline

the sample
300400

Yes/No
PXRD

Quantification of amorphous material by PXRD

10%
660

Diffraction
No
No

No

can be achieved by three methods:23 (1) measur-

ing the characteristic crystalline peak intensities;
(2) measuring the integrated peak areas of
the principal crystalline peaks or alternatively;
Ability to differentiate
Calibration required
Sample weight (mg)

(3) measuring the intensity of characteristic

Time of analysis

region of amorphous scattering; of physical

Phase detection

surface/bulk
amorphicity

mixtures of known crystallinity to yield a calibra-

Internal std

Destructive

tion curve which is used for further quantification

LOD/LOQ

Remarks
(min)

studies.
In the first method, the degree of crystallinity is
measured taking the area under the peaks into

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006 DOI 10.1002/jps
 QUANTIFICATION OF AMORPHOUS/CRYSTALLINE PHASES
consideration. The intensity of the X-ray scatter-
ing from the crystalline region of the sample (Ic) is
proportional to the area under the sharp peaks
above the background scattering, while the inten-
sity of the X-ray scattered from the entire sample
(Ic Ia) is proportional to the total area of the X-ray
pattern.36 The principal crystalline peaks are inte-
grated to give the area under the curve and this is
then correlated with the degree of crystallinity
by36
C Rx  100 5 metal powder, and zinc oxide.
where, Rx is the ratio of the area under the
crystalline peaks to the total area.
The amorphous content of the sample (%
disorder) can also be ascertained in similar way
from the following equation.22
% Disorder 100
X
Xa =Xc
Xa   100
6
where, X is the area under the curve measured for
the sample and Xa and Xc are the same for
completely amorphous and crystalline samples,
respectively.
By using the integrated peak intensities, the
variations in the lattice strain and particle size,
which can have significant influences on the line
shape, will not be reflected in the final result.
Other examples of quantitative analysis using
integrated peak area include digoxin,36 cefazolin
sodium,42 raffinose,43 B02669,44 b-lactam,30 a-
lactose monohydrate; b-lactose,45 cromolyn
sodium,23 acetaminophen,46 cefditoren pivoxil,47
lactose,48 microcrystalline cellulose,49 b-cyclodex-
trin,27 cephalothin,50 theophylline,51 TAT 59,20
and lactose.52
However, in certain cases the presence of over-
lapping peaks may preclude the determination of
integrated intensity. Under such circumstances,
maximum intensity of the crystalline peaks
(method 2) may be used for quantification pur-
poses provided the following conditions are met: (i)
a constant proportionality between maximum and
integrated intensity is evident; (ii) the crystallite
size is not so small as to cause particle-size-induced
line broadening; and (iii) all the analyses are
carried out under identical conditions.41
The use of an internal standard (IS) is advo-
cated, while using the maximum intensities for
quantification purposes. However, the IS should
have following properties for successful quan-
tification: (1) the selected compound must have
high-crystal symmetry so that strong, but few,
DOI 10.1002/jps
1645
diffraction peaks are produced; (2) the high-
intensity lines in the active ingredient and the IS
that are to be used for quantitative purposes
should not interfere with one another, while being
close to one another; (3) the density of the IS should
be close to that of the sample ingredients so that
homogenous mixtures can be prepared for analy-
sis; and (4) the IS must be chemically stable in the
experimental conditions.41 Examples of different
ISs, which are most commonly used in pharma-
ceutical analysis, are lithium fluoride, silicon
In the second method, a constant proportion of
appropriate IS is mixed homogenously with the
sample and thereafter the diffraction pattern is
recorded. The peak intensities of lines unique to
the sample and that to the IS are identified,
measured and the intensity ratio (intensity of
sample peak/intensity of IS peak) is used to
compute degree of crystallinity. Either the peak
height or the integrated peak intensity22 can be
taken as an indicator of intensity. By using an IS,
the problems of crystalline and amorphous overlap
and background scattering are overcome. Surya-
narayan et al.53 estimated the percent crystal-
linity in calcium gluceptate by this method
wherein the ratio of peak intensities at 2y values
of 20.18 (Ca gluceptate) and 45.08 (LiF, used as an
IS) plotted as a function of percent crystallinity of
physical mixtures was used to yield a standard
curve for further analysis. This methodology was
also used to estimate the degree of crystallinity in
various other substances using LiF as the IS like
MK-0591,54 sucrose,22 frusemide,55 indometha-
cin,56 cephalexin,21 indomethacin, sucrose,34 and
frusemide (ZnO as IS).57 The above described
methods help in estimating the crystalline content
present in the sample, from which the amorphous
content of the sample can be inferred. However, in
the third method the direct quantification of
amorphous content can be done using the region
where no crystalline peaks appear in the patterns
of crystalline and amorphous samples.58 In this
particular region, the diffraction is caused by the
short-range ordering in the disordered material
and hence the intensity in this region is related to
the amount of amorphous content present in the
sample and so can be used to calculate the degree of
amorphous content. This method has been shown
to be more robust and sensitive as compared to the
integrated peak intensity measurement.23,34
Recent advances in this area include the use of
whole pattern fitting to quantify the amorphous
content in the sample, which requires that the
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
1646 SHAH, KAKUMANU, AND BANSAL
peak position and peak shapes for a specific phase
be consistent. To achieve this, a special poly
capillary optic is used which has distinct advan-
tages over the BraggBrentano Optics with
respect to sample displacement artifacts. This
new approach employs the diffractograms of the
100% crystalline and 100% amorphous forms to fit
the diffractogram of the unknown sample. Since
the information from the amorphous form in the
mixture is used, the detection limit may be pushed
down to 1%, which few analytical techniques can
achieve. In addition this method also minimizes
the traditional problems of preferred orientation
and overlapped X-ray lines.59
The sources of error while measuring the cry-
stalline/amorphous content by PXRD include41:
(1) preferred orientation which can be reduced
through grinding and/or sample back packing;
(2) measurement of line intensitythe advan-
tages of estimating peak intensity over maximum
intensity have already been mentioned earlier in
this section; (3) micro-absorption due to absorp-
tion onto crystalline faces; (4) maximum accepta-
ble particle size which is to be optimized in order
to minimize broadening effects and avoid pre-
ferred orientation; and (5) statistical errors
which can be minimized by employing a long scan
time. The other possible sources of error are
primary and secondary extinction effects, inade-
quate sample thickness, and finally instrumental
errors.
The principal disadvantage in using PXRD
method is that, it may not be suitable for samples
with complex scattering pattern, since the isola-
tion of the peaks may be difficult and the separa-
tion of the amorphous scattering from the total
diffraction could be ambiguous.30 Also some low-
intensity peaks may exist in the background
because of the effect of residual solvent in the
sample, which may interfere with the interpreta-
tion of results.60
Thermal Techniques
Analytical techniques based on thermal energy
principle have been widely employed to charac-
terize amorphous pharmaceutical systems.18
Crystallization from the amorphous state can be
induced by the thermo-analytical techniques to
produce an exothermic change whose magnitude
is then quantitatively related to the extent of
crystallization occurring. This can then be used to
determine the initial percent crystallinity of a
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
partially amorphous sample provided total crys-
tallization is known to occur.22,32
Differential Scanning Calorimetry (DSC)
DSC is widely used for investigating the phase
behavior of pharmaceutical solids, including quan-
tification of the amorphous content.61 For the
latter, several methodologies based on the instru-
ment type, measured parameters, and experimen-
tal conditions are utilized. Generally employed
DSC techniques include: (i) conventional DSC; (ii)
modulated temperature DSC (MTDSC); and (iii)
hyper or high-speed DSC (HSDSC)62 all of which
are discussed in proceeding sections.
Conventional DSC. Conventional DSC is based on
linear heating rate of the sample through its
melting point. A typical DSC scan of an amorphous
or mixture of amorphous and crystalline material
shows a glass transition endotherm, crystalliza-
tion exotherm, and fusion endotherm.22
The presence of amorphous phase is distinctly
seen as a glass transition endotherm, although
this might be difficult to observe for low levels of
amorphous state. The amorphous content present
recrystallizes to give the crystallization exotherm
and the resultant crystalline state along with the
preexisting crystalline content, if present, melt
together to give the melting endotherm. For
quantification of the amorphous content, a cali-
bration curve is prepared initially using 100%
amorphous and crystalline standards and their
physical mixtures, and their respective heats of
crystallization or enthalpies of fusion. The 100%
amorphous standard is prepared in situ in the
DSC. The amorphous content in a given sample
can then be estimated either from the heat of
crystallization22,63 or enthalpy of fusion23,64,65 of
the sample using the following equation.66
Xc % DH=DH0 =100 7
Where, DH is the enthalpy of fusion of the sample
and DH0 is that of the 100% crystalline standard.
The amorphous content can also be calculated
without preparing the calibration curve, using the
following equation.67
DHfamor
Amorphous content 8
DHfcry
where DHfamor is enthalpy of fusion of amorphous
fraction and DHfcry is the heat of fusion at the melt
for the purely crystalline material. Or from the
DOI 10.1002/jps
 QUANTIFICATION OF AMORPHOUS/CRYSTALLINE PHASES 1647

following equation68 is the enthalpy of fusion of pure crystalline

DHcr Tcr 1

DSC  T 1
a
9
DHm cr
where,
DSC is the amorphous fraction of the
sample, DHcr(Tcr) is the enthalpy of crystalliza-

tion at crystallization temperature, DHm Tcr is
the enthalpy of melting of purely crystalline state
at the crystallization temperature and a is the
fraction of the amorphous content that does not
crystallize upon heating.
Another approach of determining the percen-
tage crystallinity of a sample utilizing DSC with-
out preparing calibration standards involves
subtraction of the heat of crystallization from the
heat of fusion which will yield the amount of heat
due to the initial crystallinity of the sample, and
dividing this value by the enthalpy of fusion of the
pure crystalline sample and the sample mass will
yield the percentage of initial crystallinity.69,70
DHf
DHc
% Crystallinity 10
DHfcry  m
where DHf is the heat of fusion of the sample, DHc
is the heat of crystallization of the sample, DHfcry
sample, and m is the sample mass.
Modulated Temperature DSC (MTDSC). In
MTDSC, a sinusoidal wave modulation is super-
imposed over the conventional linear (or isother-
mal) heating or cooling temperature program.
MTDSC is based on the same theory as conven-
tional DSC, in which the heat flow signal is a
combination of the specimen heat capacity Cp,t
(heat-rate dependent component) and of any
temperature dependent, often irreversible,
kinetic component.71
Quantification of amorphous content of a sam-
ple using MTDSC is based on the measurement of
heat capacity jump associated with the amorphous
phase glass transition by preparing a calibration
curve based on the heat capacity jumps of physical
mixtures of known crystallinity as shown in
Figure 1.71
AmpMHF
Cp KCp  11
AmpMHR
where K(Cp) is the heat capacity constant,
AmpMHF and AmpMHR are the amplitudes of

Figure 1. Heat capacity (Cp) jumps, recorded on various known mixtures of

amorphous and crystalline forms of a developmental compound using modulated
temperature differential scanning calorimetry (MTDSC). (Reproduced with permission
from Reference 71.)

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
1648 SHAH, KAKUMANU, AND BANSAL
modulated heat flow and heat rate, respectively.
Cp; theoretical
KCp 12
Cp; measured
However, for precise heat capacity measurements
several points like the thickness of the sample bed
in sample pan, the thermal contact resistance
between the sample and the sample pan, and
the thermal contact resistance between the sample
pan and the base plate of the apparatus have to be
considered in order to get reliable results.72
Hyper DSC. High-speed or high-performance
DSC (hyper-DSC) has been reported to have high
sensitivity while operating at extremely high-scan
rates, in the range of 1005008C/min. The sensi-
tivity of the DSC response peak increases sub-
stantially; as the scan rate is increased because
increased scan rate leads to higher heat flow.
Higher heating rates show highest sensitivity,
thus helping in identifying or determining small
quantities of amorphous content present in the
sample. The amorphous content is calculated
either from the height of Tg61 or from the specific
heat measurements.62 There is obviously a great
advantage in this as the amorphous form is not
thermodynamically stable; hence quick detection
minimizes the chance of it being lost during the
experiment.61 However, this technique is still to
find wider application in the pharmaceutical field
as in most cases, quantification is done utilizing
conventional DSC only.
The use of hyphenated techniques for quantifi-
cation purposes has been necessitated due to the
constraints of the traditional techniques in case of
quantification in formulation matrix systems,
wherein there is a possibility of drugexcipient
interaction. Recently the use of an HPLC-DSC
technique was reported for quantification of
amorphous content in a sustained release formu-
lation (NIC-LA1) of nicardipine (NIC)73 in which
the quantity of amorphous NIC is calculated as the
difference between the quantity of total NIC
determined by HPLC and the quantity of crystal
NIC determined by conventional DSC. This
method was confirmed to have sufficient accuracy
and reproducibility for estimating the content of
amorphous NIC in NIC-LA1 thereby ensuring the
safety and efficacy of the formulation.
Isothermal Microcalorimetry (IMC)
IMC was one of the earliest methods to be
explored as a tool for quantifying amorphous
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
content in powders.74 Recent reports39,7477 have
demonstrated the remarkable sensitivity of IMC
in the study of crystallinity changes especially in
the assessment of amorphous content. IMC and
gravimetric methods are the only two methods
wherein the amorphous state is measured pre-
dominantly without the interference of crystal-
line response.39 This method is based on the
principle that almost all physical and chemical
processes are accompanied by a heat change78
and so the energy changes associated with the
recrystallization process of amorphous powders at
certain relative humidities (RH) and tempera-
tures or in the presence of organic vapors can be
monitored to ascertain the amorphous content of a
sample.
The micro calorimetric approach involves expo-
sing the powder sample containing the amorphous
phase to vapor, thus encouraging recrystallization
of the powder.79 This occurs when the critical
moisture content has been reached which is
sufficient to lower the Tg below the operational
temperature.45 The lowering of Tg leads to increa-
sed molecular mobility facilitating crystallization.
The simplest approach of studying the amorphous
content in a powder is to seal the powder in a glass
ampoule with a small tube containing saturated
solution, which will yield a definite relative
humidity.39 This ampoule is then held in a sink
under isothermal conditions such that any process
occurring in the ampoule can be monitored either
by gain or loss of heat to the sink.74,78 The output is
recorded in the form of rate of change of heat (i.e.,
power) as a function of time.45 The conversion of
amorphous to crystalline form is detected as an
exothermic heat flow, the integrated peak area of
which is directly proportional to the amorphous
content.80 Area under the power-time curve is
compared to that of an amorphous standard for
quantification purposes.81
The degree of amorphous content for any
sample can be calculated from the following
equation:
% Degree of disorder 100  Q
blank=Qa
13
where Q denotes the heat output for the test
sample and Qa denotes the heat output for a
totally amorphous sample provided the same RH
is used and the sample loading is identical or
normalized. The blank response relates to the
output obtained by loading the cell into the
calorimeter.74
DOI 10.1002/jps
 QUANTIFICATION OF AMORPHOUS/CRYSTALLINE PHASES
In order to obtain a quantitative value for
degree of amorphous content in a sample, the
loading process must be standardized.74 The
important variables to be considered include:
(i) the temperature differential between outside
environment and the calorimeter, before loading
into the equilibration position and (ii) the length of
time at the equilibration point before lowering into
the measuring site.
A typical response for amorphous lactose equili-
brated at 258C 75% RH (saturated sodium chloride
solution) is shown in the Figure 2.39 In this figure,
the initial slow peak (phase 1) for the amorphous
sample is a composite of vapor-phase wetting of the
powder (exothermic), collapse, and generation of
humid air from saturated salt solution (endother-
mic).39,45 Since the initial peak directly starts with
an exothermic heat flow, it indicates that the
process has started before it could be monitored by
the microcalorimeter due to necessary tempera-
ture equilibration period.45 If the water vapor were
generated remote from the measuring site, the
wetting response would be very large and would
potentially obscure the recrystallization peak,
which is the main location of interest.74 The large
exothermic peak (phase 2) represents the recrys-
tallization of the amorphous content present in the
sample.45 This peak is very sharp showing rapid
decline which indicates the crystallization process
is extremely cooperative and it occurs simulta-
neously throughout the entire sample bed only
Figure 2. Typical crystallization response for amorphous lactose at 75% RH and 258C
in an isothermal calorimeter. (Reproduced with permission from Reference 39.)
DOI 10.1002/jps
1649
after the entire sample is saturated with water
vapor.74 The curve shows a phase 3 until the signal
reaches zero. This phase was earlier thought to
represent the expulsion of water vapor from the
powder sample because of recrystallization and
condensation of this vapor, but the heat flow due to
these processes would cancel out each other or give
a small endotherm.45,82,83 This phase is now
thought to be due to the mutarotation from b to
a-lactose.45
However, the results obtained are significantly
influenced by various factors like: (1) powder load
variations;45,74 (2) source of humidity; (3) varia-
tions in available humidity;45,74,78,84,85 (4) changes
in available surface of saturated salt solutions;74
(5) changes in the proportion of the organic
vapors;80,86 (6) effect of prestorage of the sample
at a particular humidity;74 (7) changes in operatio-
nal temperature;39 (8) effect of time and pressure
employed during micronization of sample;45,74,80,8691
and (9) integration methodology87 and hence these
need to be optimized in order to get reproducible
and reliable results.
The systematic study of different variables
involved in IMC lead to following conclusions:
(i) larger sample loads increased the lag period,
that is, the time after which recrystallization
occurs;45,74 (ii) higher RH conditions lead to faster
crystallization sometimes even before equilibra-
tion;45,74,78 (iii) the availability of greater surface
area of saturated salt solutions lead to faster
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
1650 SHAH, KAKUMANU, AND BANSAL
crystallization;74 (iv) increase in both milling time
and pressure lead to increase in the amorphous
content which was reflected as an increase in the
time to peak, the peak height and the area under
the curve.74,77,78
One of the important problems encountered
while using IMC, is that samples with lower
degree of amorphous content tend to crystallize
faster than those with greater degree of amor-
phous content, often before equilibration of the
system, leading to difficulty in interpretation of
results. To delay crystallization event, a greater
sample size of perhaps several hundred milligrams
is required, which can be a limitation in certain
cases, for example, when dealing with a new drug
substance.91 The usage of this technique is at
present limited to amorphous solids that crystal-
lize spontaneously under certain RH and tempera-
ture conditions or organic vapors.60
Solution Calorimetry (SC)
Solution calorimetry, also known as isoperibol
solution calorimetry, is a thermal technique in
which the temperature change produced by a
chemical or physical interaction during mixing of
two solutions or of a solid or a liquid in a constant
temperature environment is monitored as a
function of time.60 It was introduced in the
pharmaceutical field by Pikal et al.30 for quanti-
tative assessment of crystallinity of pharmaceu-
ticals. Provided the energy difference between
crystalline and amorphous states is large, the
degree of crystallinity obtained by this technique
is more precise, less subject to artifacts and less
ambiguous than the crystallinity data derived
from X-ray diffraction.30
The conceptual basis of SC can be expressed by
the following one-step reaction:60
S L ! SL1 14
where S and L are the amount of free substrate
and ligand, respectively.
In this equilibrium system, the standard
enthalpy change is defined as DHo and the total
heat effect Q is related to the moles of SL1 produced
and DHo as
Q SL1  DH o 15
Here SL1 is the total sample amount used and
DHo is the heat of solution to infinite dilution in
any fixed solvent L.
This concept can be extended to the determina-
tion of crystallinity in pharmaceuticals because of
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
energy differences and subsequent differences in
the heats of solution of crystalline and amorphous
forms. This technique has been shown to be
sensitive enough to monitor the changes in crystal-
linity induced due to processing, which PXRD and
DSC failed to do. Enthalpy of solution increases in
linear fashion with decreasing amorphous con-
tent. This is because of the reason that the
crystalline material is at the lowest energy state,
and the interactions within the crystalline struc-
ture are stronger than the solvation process,
resulting in an overall endothermic heat of solu-
tion, whereas amorphous form being at higher
energy level, shows exothermic response.60 SC
works as a bulk technique, in which both responses
for the amorphous and crystalline material are
measured and hence there is a need for substantial
difference in the heats of solution of the two if small
amounts of amorphous materials are to be
detected.39 Thus the sensitivity of the technique
depends upon the difference in enthalpy of solu-
tion between the amorphous and the crystalline
forms, with larger difference leading to higher
sensitivity.92
The principle of the technique is to create a
system within which very small enthalpy changes
in a liquid system due to dispersion or dissolution
may be recorded as a function of time.92 In this
technique, known amount of a solute (solid or
liquid) and a suitable solvent are housed together
in the reaction vessel and equilibrated to the
temperature at which reaction takes place. The
solute is sealed in a small fragile glass ampoule,
which is immersed into the solvent. The glass
ampoule prevents dissolution until thermal equi-
librium has been established. Following equilibra-
tion the glass ampoule is broken, thus initiating
the reaction and allowing the enthalpy of solution
to be measured.92 The heat flow signal is mon-
itored as a function of time. By integrating the heat
flow curve over a specific interval of time, the heat
evolved or absorbed is obtained.63 In practice, the
crystallinity is determined by a linear relationship
between heats of solution and crystalline content
of physical mixtures of crystalline and amorphous
standards in different ratios. It can also be directly
calculated by using the following equation:93
 
DHso
DHao
Pc 100 16
DHco
DHao
where, DHso , DHco and DHao are the heats of
solution to infinite dilution (in any fixed solvent)
of the sample, the 100% crystalline standard, and
the 100% amorphous standard, respectively.
DOI 10.1002/jps
 QUANTIFICATION OF AMORPHOUS/CRYSTALLINE PHASES
Calibration of the instrument is performed at
each time of determination. The calibration reac-
tions normally used include Tris in 0.1 M HCl,94
KCl, or NaCl in water,95 sucrose or propan-1-ol in
water.96 In addition to range of possible calibration
materials, there are a number of different methods
available to determine the enthalpy of solution
from the experimental data provided by the
calorimeter like: (i) the RegnaultPfaundlers
method;97 (ii) a graphical extrapolation based on
the Dickinson method;97 or (iii) an integration-
based method.98 The calibrants used and the
methods used to calculate the enthalpy of solution
significantly affect the results and hence should be
properly selected to yield reproducible results.99
SC was found to facilitate the comparison of
crystallinity of the b-lactam antibiotics with their
chemical stability.93 It could also detect and
quantify the amorphous contents of unmicronized
and micronized drug100 as well as examine the
effect of different grinding times101 or different
methods of preparation of amorphous forms,63
when XRD and DSC failed to do so.
Sometimes, erroneous results are obtained
when SC is used because of difficulty in interpret-
ing the data as the net measured response for
enthalpy of solution is a sum of numerous compo-
nents, viz. wetting of powder, dissolution (disrup-
tion of bonding between solid molecules and
formation of bonds between solute and solvent),
rearrangement of bonding within solvent to
accommodate solute.60,102 Also choice of solvent
is critical to the application of this technique. The
chosen solvent must dissolve the drug relatively
rapidly and at least wet the rest of the components
in the system.60 The heat produced during the
dissolution process needs to be calibrated by a
known amount of heat. This correction can be
calculated accurately over only a limited time
period. This limits the isoperibol method to experi-
ments which are complete within 12 h.103 The
sensitivity of the method is also material depen-
dent, for the energy difference between amorphous
and crystalline forms may vary depending on the
solid forms of the sample.
In a recent study, it was shown that the
complete dissolution of the sample in the chosen
solvent was not necessary. This is beneficial as it is
not always feasible to find a solvent in which the
sample is freely soluble. An excellent linear corre-
lation was observed between the reaction enthalpy
and the amorphous content of the lactose sample
(physical mixtures) in MeOH in which it was
poorly soluble.104 Hence, it is now possible to
DOI 10.1002/jps
1651
utilize SC for quantification of amorphous content
of a drug, which is slightly soluble in the selected
solvent.
The USP has now recommended SC as a
technique for determining crystallinity of phar-
maceuticals.102,105 As compared to IMC and DVS,
SC is more appropriate if there is any serious fear
that the amorphous material cannot be accessed
by the vapors, or if there are no suitable vapors to
induce crystallization response.39
Gravimetric Techniques
Vapor sorption by amorphous and crystalline
pharmaceutical materials is usually quite differ-
ent and thus can be used to precisely distinguish
between the two.30 Typically, crystalline materi-
als adsorb vapors in small quantities at their
surfaces, or take larger stoichiometric quantities
to form solvates. In contrast, the amorphous
materials absorb vapors in relatively larger
amounts.18
Dynamic Vapor Sorption (DVS)
Water sorption or gravimetric techniques have
been extensively used in the study of many
amorphous and partially amorphous powders.39
It is a useful method for standardizing the
amorphous content either as a single component
or in combination.106 Dynamic vapor sorption
(DVS) is based on the concept of exploitation of
crystallization of amorphous materials with
changes in humidity, with consequent expulsion
of water. Extent of water sorption and desorption
is related to the amorphous content of the
sample.106 DVS works simply by detecting the
crystallization response for the amorphous mate-
rial, with little or no interfering response form the
crystalline component.39
The gravimetric studies are usually conducted
in a humidity-controlled microbalance system.
The sample is loaded on one side of a single or
twin pan balance, and the system is programmed
for measurement of sorption and desorption at
particular humidity and temperature.81 A typical
moisture sorption isotherm showing the changes
in mass at different relative humidities is pre-
sented in Figure 3.105 However, the moisture
sorption isotherms cannot be used as such for the
quantification of amorphous content as the moist-
ure absorbed by the amorphous regions as well as
that adsorbed onto the surface will contribute to
the total water sorbed by the sample.105
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
1652 SHAH, KAKUMANU, AND BANSAL
Figure 3. Moisture sorption isotherm of amorphous
and crystalline forms of a developmental candidate.
(Reproduced with permission from Reference 105.)
Quantification of amorphous content in a-
lactose monohydrate has been reported using
DVS, in which the sorption and desorption
sequence was run four times. Higher weight
increase was observed after the first sorption
cycle, which was attributed to the water being
sorbed onto the amorphous regions as well as being
adsorbed onto the surface of the crystalline
material. All the other cycles showed superimpo-
sable responses, which indicated the water being
adsorbed onto the surface of the crystalline
material. The increase in the weight from the
starting point of the first sorption run to the end of
the first desorption gave a good correlation with
the amorphous content present.88,89
Materials, which are not freely soluble in water,
are usually investigated using nonaqueous vapors
to induce crystallization.88 This is because the
hydrophobic drugs when exposed to water vapor,
may not crystallize readily as the water vapor
leads to surface crystallization which is self-limit-
ing and hence prevents further water access,107
whereas the nonaqueous vapors have higher
access to the amorphous regions and can be used
to induce crystallization. Mackin et al.88 applied
this principle to quantify low levels of amorphous
content in lactose. The method rationale was
to measure the acetone uptake (combination of
acetone adsorbed onto surface and absorbed into
amorphous regions) of the crystalline/amorphous
sample at 30% relative pressure, recrystallize the
sample at 85% relative pressure, and then mea-
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
sure the acetone uptake (adsorption of acetone
onto surface of 100% crystalline solid) of the resul-
tant crystalline sample at 30% relative pressure
again. By subtracting the second mass uptake of
acetone at 30% relative pressure from the initial
uptake value, the amount of absorbed acetone was
calculated, which was directly proportional to the
amorphous content of the sample (r2 0.999).
Recent advances in the analysis of pharmaceu-
ticals have made possible the use of hyphenated
techniques. One such technique is used in the
quantification of amorphous systems where a
NIRS probe is housed beneath the sample pan of
DVS. Here NIRS has been used to assess the
amorphous content of samples when dry, and the
DVS data is used to assess the amorphous content
by calculation of the extent of water sorption and
desorption. The area under the positive peak of the
dm/dt plot (Fig. 4) is the quantity of water sorbed
during the sorption run whereas the area under
the negative region of the dm/dt plot is the quantity
of water desorbed. The mass of the sorbed water
gave a good correlation (r2 0.929) with the
amorphous content of the sample.104
In gravimetric analysis, a clear sign of presence
of amorphous material in a sample is the loss of
mass on exposure to increasing relative humidity.
Conversely, the absence of mass loss is interpreted
as the absence of a crystallization event. However,
the absence of a crystallization event would not
imply the absence of the amorphous content, but
only that the sample did not crystallize. As, for
example, raffinose is able to form different
hydrates depending upon the conditions of tem-
perature and humidity.89 At 75% RH and 258C, the
sample seems to crystallize to the tetrahydrate as
observed using NIR spectroscopy. The mass
required to form the hydrate was equivalent to
that which was absorbed. If mass change alone
were relied upon to detect crystallization of
samples for which multiple hydrates can form,
then it would be possible to miss the crystallization
event. These data show the clear value of using
more than just gravimetry as a method to assess
onset of crystallization.
Spectroscopic Techniques
Spectroscopic methods are nondestructive and
can be used alone or in conjunction with other
solid-state techniques (TGA, microscopy, DSC,
XRD) for quantitative analysis of pharmaceutical
solids. Once suitable spectral features, which
arise from observed crystallographic differences,
DOI 10.1002/jps
 QUANTIFICATION OF AMORPHOUS/CRYSTALLINE PHASES 1653

Figure 4. Rate of change of mass (dm/dt) against time for a 5% w/w amorphous lactose
sample observed using dynamic vapor sorption (DVS). (Reproduced with permission from
Reference 104.)

are identified, they can be used to develop
methods for the quantitative analysis of one phase
in the presence of the other.108 One advantage of
spectroscopic methods over diffraction methods
for quantitative analysis is these methods are
often superior for the quantitative analysis of
crystallinity, since Raman, IR, or ss-NMR spectra
of amorphous phases give specific, albeit broa-
dened signals. The nondestructive nature of
spectroscopic methods renders these techniques
superior to thermal methods of analysis in most
cases.40
Vibrational Techniques
In contrast to PXRD, which probes the orderly
arrangement of molecules in the crystal lattice,
vibrational spectroscopy probes differences in the
influence of solid state on the molecular spectro-
scopy. Of the spectroscopic methods which have
been shown to be useful for quantitative analysis,
vibrational (mid-IR absorption, Raman scatter-
ing, and NIR) spectroscopy is perhaps the most
amenable to routine, online, offline, and quality-
control quantification.40 In developing a quanti-
tative method based on vibrational spectroscopy,
measures must be taken to ensure homogeneous
sample mixing, particle size, and instrument
variability and reproducibility.109
Infrared Spectroscopic Technique (IR). IR techni-
ques reflect significant spectral differences
between crystalline and amorphous phases and
hence are used to quantify the crystalline content,
as the intensity of the vibrational bands is directly
proportional to the concentration of the concerned
phase.21,24,49,66 Infrared procedures for measuring
the degree of crystallinity are based upon the
measurement of intensity of a peak, which is
characteristic of the crystalline state with refer-
ence to a peak, which is independent of the crystal
state of the substance.36
In case of cefazolin sodium42 the intensity of the
band at 1760/cm remained invariant in the crystal-
line and amorphous forms whereas the band at
1542/cm as indicated by the area under the curve

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
1654 SHAH, KAKUMANU, AND BANSAL
decreased as the percentage of the amorphous
form increased. The percentage ratios calculated
for standard samples of physical mixtures when
plotted against the percentage crystallinity were
reported to have produced a good correlation of
0.993.
Nakai et al.49 studied the effect of grinding on
the crystallinity of microcrystalline cellulose
(MCC) by infrared technique. In this study, it
was seen that the hydroxyl groups of the cellulose
molecules in amorphous region were rapidly con-
verted to deuteroxyl groups upon treatment with
deuterium oxide vapor, whereas the deuteration
was very slow in crystalline region. The compar-
ison of the areas of absorption band due to hydroxyl
and deuteroxyl groups leads to determination of
the ratio of crystalline region to amorphous one for
MCC.
Similarly the effect of grinding on the crystal-
linity of cephalexin was also studied by Otsuka and
his coworkers.21 Here it was seen that the peak
intensities of absorption bands of b-lactam uc 0 at
1750/cm and that of carbonyl uc 0 at 1580/cm of
cephalexin changed with increasing grinding
time. The degree of crystallinity (X00cr ) of a sample
was obtained by substituting the absorbance ratio
into Eq. 17
X00cr fA1580 =A1750
0:7954g=0:3564  100
17
24
Kaneniwa and his coworkers used this equation
to investigate the relationship between com-
pression parameters (stress and loss energy)
and the physicochemical parameters (the de-
hydration and decomposition points and the
crystallinity).
Near Infra-Red Spectroscopy (NIRS). NIRS is fast
becoming an important technique for pharmaceu-
tical analysis as it is simple, nondestructive
technique that requires no sample preparation,
especially if used with diffuse reflectance option.
Apart form identification and quantitative analy-
sis, the method can also be used to characterize
particle size, polymorphism, and control technolo-
gical procedures like blending, coating, and
drying.110 A variety of chemometric and statistical
techniques have been used to extract pharmaceu-
tically relevant information from raw spectro-
scopic data. Calibration models generated by
multiple linear regression (MLR) analysis, princi-
pal component analysis, and partial least squares
regression analysis have been used to evaluate
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
various parameters.111 The choice of the calibra-
tion method is dictated by the nature of the sample
and the number of components to be simulta-
neously determined.112
Usually quantification of crystallinity is per-
formed using the first106 or second derivative
spectra.34,112,113 First derivative spectra enables
enhancement of peak resolution.106 Second deri-
vative spectra helps to minimize the well-known
effects of particle size and variable scattering on
NIR radiation.34 Such a treatment also has the
added benefit of normalizing the baseline (exclu-
sion of the upward baseline shift) and sharpening
spectral features.
In case of quantification of miokamycin by
Blanco et al.,114 it was found that the NIR spec-
tra of the crystalline and amorphous form of
miokamycin showed strong differences to enable
detection of the crystalline form both in the
amorphous phase and in the pharmaceutical
preparation. The best results were obtained with
the first derivative mode over the wavelength
range 11001800 nm.
Gombas et al.113 utilized second derivative
spectra for quantification of crystallinity of lactose,
to overcome the effects of particle size and variable
scattering on the NIR radiation as shown in
Figure 5. Using the second derivative spectra,
multiple linear regression was performed at dif-
ferent wavelengths and crystallinity was deter-
mined in the physical mixtures of crystalline and
amorphous lactose.
Otsuka et al.115 established a chemoinfometri-
cal method based on FT-NIR spectroscopy to
evaluate degree of crystallinity of indomethacin
(IMC) and compared the chemoinfometrical
method with the conventional powder X-ray dif-
fraction method. They found that the results were
comparable and the FT-NIR technique was more
accurate with a mean accuracy of 2.46%.
Water sorption can significantly affect NIR mea-
surements. Hogan et al.104 have used a combina-
tion of NIRS and DVS, where the NIR probe is
housed immediately below the sample pan of DVS.
This allows both the drying of samples to suitable
endpoint, enabling elimination of water-related
interference in NIRS and the humidification of
samples with the ability to monitor the consequent
changes in physical form with the NIRS. There are
obvious advantages to having both the techniques
operating in combination because an immediate
check of the NIR data can be obtained from the
DVS sorption response. This hyphenated techni-
ques needs to be explored further.
DOI 10.1002/jps
 QUANTIFICATION OF AMORPHOUS/CRYSTALLINE PHASES
Figure 5. Second-derivative NIR spectra of absorbance of mixtures of lactose with
varying crystallinity. (Reproduced with permission from Reference 113.)
Fourier-Transform Raman Spectroscopy (FTRS).
Vibration is IR active when there is a change in
molecular dipole during vibration. On the other
hand, vibrational mode is Raman active when
there is a change in polarizability during vibra-
tion.116 Generating data complementary to that
from infrared spectroscopy, FTRS provides unique
capabilities like: (i) minimal sample preparation,
thereby preventing the likelihood of inducing
any phase changes; (ii) rapid collection of data;
(iii) nondestructiveness; and (iv) minimum inter-
ference of water with the vibrational modes from
most drugs.117 A special advantage of FTRS lies in
the fact that relatively nonpolar components are
highly Raman active, and most excipients are
poor Raman scatterers, making FT-Raman an
excellent method for dosage form analysis.40
Raman spectra in 500525/cm spectral range is
useful for studying low-frequency lattice vibra-
tions, ring vibrations, heavy atom fundamental
vibrations, and pure rotational spectra for gas-
eous samples.116
Correlation curve for indomethacin sample was
prepared for quantification using carbonyl peaks
at 1698/cm for crystalline sample and at 1680/cm
for amorphous sample.118 As the two peaks were
not completely resolved as a consequence of
broadening of the carbonyl peak in the amorphous
DOI 10.1002/jps
1655
spectrum, the contribution of the amorphous peak
to the crystalline peak intensity and vice versa was
taken into account. After appropriate calculations,
a linear correlationship (r2 0.9996) was observed
between the percentage crystallinity and the peak
response, with LOD and LOQ values of 0.6% and
2.0%, respectively.
Chan et al.119 used this technique to measure
the initial crystallinity and changes in crystal-
linity of constituent powder materials upon
storage and exposure to different humidity
conditions of various spray dried formulations
containing salmon calcitonin (sCT) and Manni-
tol. The crystallinity of freshly made formula-
tions was determined by subtracting the
contributions of sCT and the various Mannitol
polymorphs in the formulation to the Raman
spectra.
Other advantages of Raman spectroscopy
include: (a) it provides a direct probe of molecular
structure of the compound; (b) samples can also be
analyzed directly in containers such as glass/
plastic bottles and blister packs thereby involving
minimal handling; and (c) use of fiber optics
Raman spectroscopy can enable process analysis
in on-, at-, and in-line applications, and in a
loading/receiving dock environment can be done
for identity testing.116
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
1656 SHAH, KAKUMANU, AND BANSAL
Sources of errors in measuring the percentage
crystallinity of a sample by FT-Raman spec-
troscopy can be the laser power, instrument
variability, sample homogeneity, and overall
method error.118 Sample heating is a well-known
problem associated with laser Raman spectro-
scopy. Increase in laser power, or scanning of same
spot 10 times consecutively, can contribute sig-
nificantly to sample heating, and possible phase
changes. Shaking the sample intermittently while
taking repeat scans can reduce errors due to
sample inhomogeneity. Also, Raman laser excites
only a very small portion of sample (1 mm in
diameter) in 13 mm diameter sample cup or 5-mm
glass tube, which may not be a true represen-
tative of the entire sample, if sample is not
homogeneous.
In the recent reports,120 a charge coupled device
(CCD) Raman detector was used for quantification
of crystallinity of lactose, instead of a FT detector
to allow for shorter measurement times. Although
there is a danger of interference due to fluores-
cence, this is unlikely to hamper quantification in
pharmaceuticals, as pure pharmaceuticals are
nonfluorescent. Here the ratios of bands unique
to amorphous and crystalline forms were used to
quantify the amorphous content. As shown in
Figure 6, the bands centered at 470 and 400/cm
representing monohydrate and amorphous forms
of lactose varied smoothly with the changing
component ratio. The total measurement time
Figure 6. Raman spectra of mixed amorphous/mono-
hydrate samples. The band at 470/cm increases with
increasing monohydrate content: 0%, 20%, 40%, 60%,
80%, and 100%. (Reproduced with permission from
Reference 120.)
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
was reduced to 10 min as compared to few hours
required with the FT detector.
Solid State NMR (ss-NMR)
High-resolution 13C ss-NMR spectra are obtained
using proton decoupling and magic angle spin-
ning (MAS) and sensitivity enhancement is
achieved by cross-polarization (CP). 13C ss-NMR
has the advantage of being a nondestructive test
method that provides information about the
structure of the material.121 Like in any other
one-dimensional NMR method, it is possible
to relate straightforwardly the integral of the
CPMAS NMR signal to the number of 13C atoms
involved, provided relaxation rates, Hartmann
Hahn conditions and cross-polarization rates are
properly investigated for each species in the
sample.122 In cases where the reference spectra
of the individual constituents are unavailable,
quantitative estimation of defects, amorphous
contents, or mixed phases by NMR can be done
based on the comparison of the integrated inten-
sity of two separate lines in the spectrum.123,124
According to Ek et al.,124 peaks unique to
amorphous and crystalline samples were inte-
grated and a crystallinity index for microcrystal-
line cellulose was determined in the following way
CrI a=a b 18
where a is the integration of peaks between 86
and 93 ppm and b is the integration of peaks
between 8086 ppm.
However, this type of analysis can sometimes be
tricky especially if the two lines under scope are
overlapping and cannot be easily deconvoluted.125
These difficulties can be overcome by resorting to
other independent measurements like T1 or T1r
relaxation times of 1H or 13C, relying on the
expected difference in the mobility of amorphous
and crystalline regions.126128
Lefort et al.68 studied the efficiency of 13C
CPMAS NMR for the determination of amorphous
and crystalline fractions in molecular materials
taking trehalose as an example. In this case, the
reference spectra of crystalline and amorphous
trehalose were available; hence a simple and
precise deconvolution method was used for quan-
tification. The amorphous and crystalline spectra
along with the spectra of their physical mixtures
are shown in Figure 7.
The asymmetric broadening of the NMR line
observed in the amorphous state is due an
inhomogeneous distribution of isotropic chemical
DOI 10.1002/jps
 QUANTIFICATION OF AMORPHOUS/CRYSTALLINE PHASES
Figure 7. 13C CPMAS NMR spectra of physical
mixtures of 100% crystalline and amorphous trehalose.
(Reproduced with permission from Reference 68.)
shifts, which can be assigned to static fluctuations
of bond or torsion angles.125 In this case, it was
assumed that the NMR spectra (Si) contains only
reference species (ai and ci) and the NMR signal at
a particular frequency is proportional to the
number of carbon atoms resonating at this freq-
uency. If the sample contains a certain t amor-
phous fraction, then the normalized NMR signal
shall verify
Si tAi 1
tCi 19
where Ai and Ci represent the magnetization
signal arising from the carbon nuclei.
The amorphous fraction can then be determined
by straightforward linear regression procedure.
This method gave a good correlation between
weighed and calculated fractions in comparison
to the DSC method.
Quantification of Nicardipine (NIC) in a long
acting formula (NIC-LA) was performed using ss-
NMR as one of the quantification techniques by
Kohinata et al.73 Measurement was carried out
using the typical signals reflecting the character-
istics of crystal NIC and amorphous NIC by the
DOI 10.1002/jps
1657
peak partition method, using a crystal/amorphous
mixture (1:1) as a reference sample.
Terahertz Pulsed Spectroscopy (TPS)
Terahertz radiation lies between the IR and
microwave regions of electromagnetic spectrum
and can be defined as having a frequency of be-
tween 0.1 and 3 THz, corresponding to 3.3100/cm.
As compared to conventional far-IR spectroscopy,
TPS offers several advantages like: (i) insensitiv-
ity to thermal interference which allows measure-
ments to be made on high-temperature materials
and processes; (ii) time-resolved studies on the
subpicosecond time scale performed potentially
allowing insight into dynamic systems such as
amorphous systems; and (iii) low energy used in
TPS minimizes the risk of sample degradation.129
Responses observed in infrared and NIR spec-
troscopies are due to intramolecular vibrations
and intermolecular hydrogen bonding. Spectro-
scopy in the terahertz regime directly probes
lattice vibrations and also low-energy hydrogen
bonding vibrations.130 Therefore, the modes in
terahertz region are directly affected by the
changes in intermolecular bonding. Thus, the
spectra of different solid-state forms of a same
compound may show more pronounced spectral
changes in the terahertz regime than in the IR or
NIR region.
Quantitative analysis is performed on physical
mixtures and the raw spectra are converted into
the first-derivative spectra. The selection of spec-
tral regions for calibration is based on the largest
differences between the pure forms in the pre-
processed data as well as the largest spectral
loadings for the first factor.
Strachan et al.129,130 studied the g-crystalline
and amorphous forms of indomethacin (IM) using
this technique. As shown in Figure 8,130 the
crystalline form of IM showed distinct crystalline
peaks at 41, 50, and 66/cm, whereas the amor-
phous form showed no such peaks. The observed
signals with crystalline IM are due to intermolec-
ular vibration modes of long-range order. These
distinct crystalline peaks were used for quantifica-
tion purpose. The limit of detection using TPS was
found to be in the range of 1%5%, which
demonstrated the utility of TPS to differentiate
solid state forms of organic molecules in pharma-
ceutical settings.129
Thermally Stimulated Current Spectrometry
The thermal stimulated current (TSC) technique
is widely used in characterizing the amorphous
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
1658 SHAH, KAKUMANU, AND BANSAL
Figure 8. Absorbance spectra of crystalline (solid
line) and amorphous (dashed line) indomethacin
mixed with polyethylene powder (3:1) obtained using
terahertz spectroscopy. (Reproduced with permission
from Reference 130.)
phase of polymers131,132 by monitoring molecular
motions. Relaxations in materials take place as a
result of internal motions induced by thermal,
mechanical, or electrical energy disturbances.25
The method involves applying of an electric field
to the material at given conditions of experiment
such as temperature, in order to orient dipoles
within the molecular chains, which are sensitive to
the electric field. The temperature is then lowered
(with the electric field maintained) in order to
reduce internal motions trapping the polarized
dipoles within the material. During the reheating
of the sample, the thermal energy helps relax the
trapped molecular segments. As this occurs, a
small current is observed, corresponding to one or
more relaxations.133
Venkatesh et al.25 demonstrated the applicabil-
ity of TSC for the detection of low levels of the
amorphous phase in crystalline pharmaceuticals
taking carvedilol as the model drug. In TSC scans,
the glass transition event due to molecular
mobility of amorphous phase was manifested as a
peak/shoulder on the low-temperature side of the
depolarization peak of the crystalline phase. The
amorphous content can then be quantified by
estimating the area under the glass transition
curve in the spectrum of current (amps) versus
temperature (8C) for the physical mixtures. Glass
transition events measured by TSC are similar to
those measured by DSC. However, TSC offers the
flexibility of quantifying low levels of amorphous
content by polynomial curve fitting, at the same
time enhancing the response by increasing the
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
stimulus, that is,. by applying increasing electric
fields, thereby increasing the sensitivity.
Other Techniques
Density Measurement
Solid density is a physical property the value of
which is frequently required in both fundamental
and applied pharmaceutics. Although there are
exceptions, crystalline materials in general have
a higher density than their amorphous counter-
parts because the atoms in the crystal lattice are
located at a minimum possible distance from each
other. An increase in lattice disorder (decreasing
crystallinity) usually results in an increase in
volume and therefore a decrease in density.53
Hence density measurements can also be used as
an alternative technique for investigating the
crystallinity of pharmaceuticals.134
Suspension density method is one of the useful
tools for density measurement that enables to
differentiate between the samples having very
small differences in density. It also provides a
method of distinguishing between the two models
of crystallinity (one state vs. two state).53 A liquid
is chosen which has a density close to that of the
solid and which neither reacts with nor dissolves
the solid. The solid is dispersed in the liquid and
the temperature altered until the solid is sus-
pended, at which temperature, the density of the
solid is equal to that of the liquid. Since the
temperature coefficient of expansion of a solid is
generally much less than that of a liquid, the effect
of temperature on the density of the solid is con-
sidered negligible. If the simple two-state model
were valid, then on dispersion in the suspending
liquid, a partially crystalline sample would sepa-
rate into two fractions as a result of the difference
in density between the crystalline and amorphous
states. On the other hand, if the one-state model
is applicable, then progressive changes in crystal-
linity should be accompanied by gradual, prog-
ressive changes in density.53 The degree of
crystallinity of a sample can be determined by:
r
ra
% Crystallinity  100 20
rc
ra
where, r, ra, and rc are the densities of sample
under investigation, amorphous standard, and
crystalline standard, respectively.
Liquid displacement pycnometry has also been
used to determine the crystallinity of several
starch samples.18 Duncan-Hewitt et al.135 com-
DOI 10.1002/jps
 QUANTIFICATION OF AMORPHOUS/CRYSTALLINE PHASES
pared three methods of density determination,
namely displacement of a liquid, displacement of a
gas, and flotation in a liquid using adipic acid
doped with various concentrations of oleic acid.
This approach has proved useful for quantifying
low levels of disorder in crystalline pharmaceuti-
cal samples.
Density measurements by helium pycnometry
have also been exploited to quantify amorphous
solids.22 This method, when applied on powdered
samples of sucrose, was associated with large
standard deviations between measured values.
Hence, this technique was found to be less capable
of detecting low levels of disorder.
Dynamic Mechanical Analysis
Dynamic mechanical analysis (DMA) measures
the mechanical properties of a sample as a
function of temperature.136,137 The solid or semi-
solid sample is held within the instrument and
subjected to an oscillating stress, measured as a
force per unit area, Pa.137 The applied oscillating
stress results in an observable oscillating strain
(deformation) within the sample. For most DMA
instruments the oscillating stress can be describ-
ed by Eq. 21
st amax sin ot 21
The stress at time t is given by s(t), amax is the
maximum stress applied, o is the angular frequ-
ency of oscillation, such that o equals 2pf, with f
being the frequency (Hz). The ratio of the applied
stress and the deformation or strain, is defined as
the modulus in the case of elastic, usually solid,
materials. In DMA experiments, the oscillating
strain lags behind the applied oscillating stress
by a phase difference defined as d. Thus, the
modulus is complex, with an in-phase (storage
modulus) component corresponding to the sam-
ples elastic response and an out of phase (loss
modulus) viscous component. The ratio between
these parameters (E00 /E0 ), gives the damping para-
meter tan d, which is proportional to the ratio of
dissipated mechanical energy (primarily as heat)/
stored mechanical energy for each cycle. The glass
transition of an amorphous material is accompa-
nied by a large change in its mechanical proper-
ties. Thus DMA is a very sensitive technique for
the identification and characterization of glass
transitions.
In order to determine the theoretical limits of
detection,138 the DMA strength was determined
and plotted against amorphous content. The DMA
DOI 10.1002/jps
1659
strength was calculated by subtracting the base-
line response from the peak height in the tan d
signal for the nonplasticized glass transition. This
normalized value was then subtracted from 1 to
give the parameter DMA strength it normalized
the signal, removing the influence of baseline
shifts (the baseline signal was simply the tan d
signal between 30 and 408C for each experiment).
Applying this analysis, gave a theoretical limit
of detection (t-LOD) and a theoretical limit of
quantification (t-LOQ) of 2.8% (w/w) amorphous
content and 9.4% (w/w) amorphous content,
respectively, for powder-pocket DMA.
Inverse Gas Chromatography (IGC)
IGC is a vapor sorption technique in which the
powder is packed in a column and known vapors
(usually at infinite dilution in a carrier gas) are
injected. From the retention times of the probes
it is possible to assess the surface nature of
the material in the column.90 As observed with
the calorimetric and gravimetric techniques, it
can be expected that this vapor sorption approach
will also be able to detect differences in samples
due to small amounts of amorphous content.39
IGC is a highly sensitive technique and has been
used to determine the specific energies of adsorp-
tion of polar probes DGSPA , which can then be used
to calculate the basic/acidic parameter ratio KD/
KA. This parameter describes the acidic and basic
nature of the powder surface and can be correlated
with crystallinity.47 Values of KD/KA of greater
than 1 mean a basic nature on the surface of a solid,
and values of less than 1 mean an acidic nature.
Therefore, these results implied that the surface of
the intact material particle was slightly acidic
since the value of KD/KA was approximately 0.8,
and the surface of the 30 min ground material
particle was highly basic since the value of KD/KA
was approximately 2.0. This indicated that the
surface of cefditoren pivoxil powder became more
basic, or electron donating, as the grinding
proceeded.
Dissolution
It is a well-recognized fact that, for many drugs,
dissolution within the gastrointestinal tract may
be the rate limiting step to absorption; hence
improvement in the dissolution rate by means of
amorphization may enhance the bioavailability of
the drug.139142 The amorphous forms, owing to
higher molecular mobility as compared to the
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
1660 SHAH, KAKUMANU, AND BANSAL
equivalent crystalline form may have enhanced
dissolution rate and this difference can then be
used to estimate the degree of amorphous content
in a given sample. Although the amorphous form
will have a higher dissolution rate because of
high-surface free energy, there is an inherent risk
of devitrification in the dissolution fluid. How-
ever, the amount dissolved has been used to
quantify crystallinity in case of solid disper-
sions.143 Here the solid dispersion of troglitazone
and PVP K30 prepared by solvent method was
considered as 100% amorphous and was used to
prepare a regression curve after mixing with
100% crystalline drug. This regression curve was
then used to calculate the apparent crystallinity
of solid dispersions prepared by melting method.
However, the main problem encountered in this
technique is the effect of surface area if not
controlled stringently and can significantly affect
results. For controlling the surface area, the
powder is compressed; however, this may lead to
probable phase changes. Also the dissolution
medium needs to be carefully selected as the
components of the medium can influence the final
outcome. Hence necessary changes have to be
made in the experimental setup to facilitate quick
and accurate quantification.
CONCLUSIONS
Over the past two decades, the increased usage
of amorphous drugs as an approach to enhance
solubility, coupled with the development of amor-
phous excipients for improved performance of
dosage forms, has necessitated the development
of efficient methods for characterization and
quantification of the amorphous content. There
are many precise and accurate methods suitable
for characterizing amorphous pharmaceutical
materials in all their configurations, including
final dosage forms. As with any scientific method,
the use of multiple complimentary techniques,
which measure the properties most relevant to
the problem or the material under consideration
is preferred. The techniques differ in their ability
to quantify the amount of order and disorder in
partially amorphous systems.
There are numerous opportunities for further
development of quantitative methods for pharma-
ceuticals. One can expect to see more frequent
application of whole pattern approaches to
quantitative X-ray powder diffraction analysis of
pharmaceuticals. There is a great deal of interest
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
currently in utilizing analytical techniques which
enable nondestructive in situ testing of raw
materials and products in addition to online
monitoring of pharmaceutical processes. NIRS is
being widely used in this capacity. Raman spectro-
scopy in conjunction with fiber optic probes can
also be used for remote sampling and online
analysis of pharmaceutical processes. Also ss-
NMR is a promising option for discerning minute
changes at the molecular level but has not been
much explored by the scientists because of the cost
involved.
Thus, the selection of a quantification tech-
nique is based on several parameters, most
notable being the sensitivity of the technique to
subtle changes. The workers in this field need to
identify their objectives and area of interest first
and then opt for a suitable technique, for desired
application.
REFERENCES
1. Wunderlich B. 1990. Thermal analysis. London:
Academic Press.
2. Slade L, Levine H. 1995. Glass transitions and
water-food interactions. Adv Food Nutr Res 38:
103129.
3. Haleblian JK. 1975. Characterization of habits and
crystalline modifications of solids and their pharma-
ceutical applications. J Pharm Sci 64:12691288.
4. Byrn SR. 1982. Solid-state chemistry of drugs.
London: Academic Press.
5. Crowe JH, Carpenter JE, Crowe LM. 1998. The
role of vitrification in anhydrobiosis. Ann Rev
Physio 60:73103.
6. Blanchard JMV, Lillford P. 1993. The glassy state
in foods. Nottingham, UK: Nottingham University
Press.
7. Greer AL. 1995. Metallic glasses. Science 267:
19471953.
8. Jenniskens P, Blade DE. 1994. Structural transi-
tions in amorphous water ice and astrophysical
implications. Science 265:753756.
9. Kauzmann W. 1948. The nature of the glassy state
and the behavior of liquids at low temperatures.
Chem Rev 43:219256.
10. Hancock BC. 2002. Disordered drug delivery:
Destiny, dynamics and the deborah number.
J Pharm Pharmacol 54:737746.
11. Pilla S, Hamida JA, Muttalib KA, Sullivan NS.
2001. Molecular solid glasses: New insights into
frustrated systems. New J Phy 3:17.1117.18.
12. Kaushal AM, Gupta P, Bansal AK. 2004. Amor-
phous drug delivery systems: Molecular aspects,
design, and performance. Crit Rev Therap Drug
Carr Sys 21:133193.
DOI 10.1002/jps
 QUANTIFICATION OF AMORPHOUS/CRYSTALLINE PHASES
13. Yu L. 2001. Amorphous pharmaceutical solids:
Preparation, characterization and stabilization.
Adv Drug Del Rev 48:2742.
14. Angell CA. 1995. The old problems of glass and the
glass transition, and the many new twists. Proc
Natl Acad Sci USA 92:66756682.
15. Byrn S, Pfeiffer R, Ganey M, Hoiberg C, Poochi-
kian G. 1995. Pharmaceutical solids: A strategic
approach to regulatory considerations. Pharm Res
12:945954.
16. Hilden LR, Morris KR. 2003. Physics of amorphous
solids. J Pharm Sci 93:312.
17. Gupta P, Bansal AK. 2005. Spray drying for gene-
ration of a ternary amorphous system of celecoxib,
PVP, and meglumine. Pharm Dev Tech 10:257
265.
18. Hancock BC, Zografi G. 1997. Characteristics and
significance of the amorphous state in pharmaceu-
tical systems. J Pharm Sci 86:112.
19. Florence AT, Salole EG. 1976. Changes in crystal-
linity and solubility on comminution of digoxin and
observations on spironalactone. J Pharm Pharma-
col 28:637642.
20. Matsunaga Y, Bando N, Yuasa H, Kanaya Y. 1996.
Effects of grinding and tabletting on physico-
chemical stability of an anticancer drug, TAT-59.
Chem Pharm Bull 44:19311934.
21. Otsuka M, Kaneniwa N. 1983. Effect of grinding
on the degree of crystallinity of cephalexin powder.
Chem Pharm Bull 31:44894495.
22. Saleki-Gerhardt A, Ahlneck C, Zografi G. 1994.
Assessment of disorder in crystalline solids. Int J
Pharm 101:237247.
23. Byard SJ, Jackson SL, Smail A, Bauer M,
Apperley DC. 2005. Studies on the crystallinity
of a pharmaceutical development drug substance.
J Pharm Sci 94:13211335.
24. Kaneniwa N, Imagawa K, Otsuka M. 1985. Effect
of tabletting on the degree of crystallinity and on
the dehydration and decomposition points of
cephalexin crystalline powder. Chem Pharm Bull
33:802809.
25. Venkatesh GM, Barnett ME, Fordjour CO, Galop
M. 2001. Detection of low levels of the amorphous
phase in crystalline pharmaceutical materials by
thermally stimulated current spectrometry.
Pharm Res 18:98103.
26. Craig DQM, Royall PG, Kett VL, Hopton ML.
1999. The relevance of the amorphous state to
pharmaceutical dosage forms: Glassy drugs and
freeze dried systems. Int J Pharm 179:179207.
27. Nakai Y, Yamamoto K, Terada K, Kajiyama A.
1985. Relationships between crystallinity of b-
cyclodextrin and tablet characteristics. Chem
Pharm Bull 33:51105112.
28. Mullins J, Macek T. 1960. Some pharmaceutical
properties of novobiocin. J Am Pharm Assoc Sci Ed
49:245248.
DOI 10.1002/jps
1661
29. Oberholzer ER, Brenner GS. 1979. Cefoxitin
sodium: Solution and solid state chemical stability
studies. J Pharm Sci 68:836866.
30. Pikal MJ, Lukes AL, Lang JE, Gaines K.
1978. Quantitative crystallinity determinations
for b-lactam antibiotics by solution calorimetry:
Correlation with stability. J Pharm Sci 67:767
772.
31. Fukuoka E, Makita M, Yamamura S. 1986. Some
physicochemical properties of glassy indometha-
cin. Chem Pharm Bull 34:43144321.
32. Yoshioka M, Hancock BC, Zografi G. 1994.
Crystallization of indomethacin from the amor-
phous state below and above its glass transition
temperature. J Pharm Sci 83:17001705.
33. Hancock BC, Shamblin SL, Zografi G. 1995.
Molecular mobility of amorphous pharmaceutical
solids below their glass transition temperature.
Pharm Res 12:799806.
34. Seyer JJ, Luner PE, Kemper MS. 2000. Applica-
tion of diffuse reflectance near-infrared spectro-
scopy for the determination of crystallinity.
J Pharm Sci 89:13051316.
35. Hendriksen BA. 1990. Characterization of calcium
fenoprofen 1. Powder dissolution rate and degree
of crystallinity. Int J Pharm 60:243252.
36. Black DB, Lowering EG. 1977. Estimation of the
degree of crystallinity in digoxin by X ray and
Infrared methods. J Pharm Pharmacol 29:684
687.
37. York P, Grant DJW. 1985. A disruption index
for quantifying the solid state disorder induced
by additives or impurities. I. Definition and
evaluation from heat of fusion. Int J Pharm 25:
5772.
38. Grant DJW, York P. 1986. Entropy of processing:
A new quantity for comparing solid-state disorder
of pharmaceutical materials. Int J Pharm 30:161
180.
39. Buckton G, Darcy P. 1999. Assessment of disorder
in crystalline powdersA review of analytical
techniques and their application. Int J Pharm
179:141158.
40. Stephenson GA, Forbes RA, Reutzel-Eden SM.
2001. Characterization of the solid state: Quanti-
tative issues. Adv Drug Del Rev 48:6790.
41. Suryanarayan R. 1995. X-ray powder diffractome-
try. In: Brittain HG, Physical characterization of
pharmaceutical solids. New York: Marcel Dekker,
Inc., pp 187221.
42. Kamat MS, Osawa T, Deangelis R, Koyama Y,
DeLuca PP. 1985. Estimation of the degree of
crystallinity of cefazolin sodium by X-ray and
Infrared methods. Pharm Res 5:426429.
43. Saleki-Gerhardt A, Stowell JG, Byrn SR, Zografi
G. 1995. Hydration and dehydration of crystalline
and amorphous forms of raffinose. J Pharm Sci 84:
318323.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
1662 SHAH, KAKUMANU, AND BANSAL
44. Thompson KC, Draper JP, Kaufman MJ, Brenner
GS. 1994. Characterization of the crystallinity of
drugs: B02669, a case study. Pharm Res 11:1362
1365.
45. Sebhatu T, Angberg M, Ahnleck C. 1994. Assess-
ment of the degree of disorder in the crystalline
solids by isothermal microcalorimetry. Int J
Pharm 104:135144.
46. Gilani K, Najafabadi AR, Barghi M, Rafiee-
Tehrani M. 2005. The effect of water to ethanol
feed ratio on physical properties and aerosolization
behavior of spray dried cromolyn sodium particles.
J Pharm Sci 94:10481059.
47. Ohta M, Buckton G. 2004. Determination of the
changes in surface energetics of cefditoren pivoxil
as a consequence of processing induced disorder
and equilibration to different relative humidities.
Int J Pharm 269:8188.
48. Ohta M, Oguchi T, Yamamoto K. 1999. Evaluation
of solubility parameter to predict apparent solubi-
lity of amorphous and crystalline cefditoren
pivoxil. Pharm Acta Helv 74:5964.
49. Nakai Y, Fukuoka E, Nakajima S, Hasegawa J.
1977. Crystallinity and physical characteristics of
microcrystalline cellulose. Chem Pharm Bull 25:
96101.
50. Otsuka M, Kaneniwa N. 1990. Effect of grinding
on the crystallinity and chemical stability in the
solid state of cephalothin sodium. Int J Pharm 62:
6573.
51. Gubskaya AV, Lisnyak YV, Blagoy YP. 1995.
Effect of cryogrinding on physico-chemical proper-
ties of drugs. I. Theophylline evaluation of particle
sizes and the degree of crystallinity, relation to
dissolution parameters. Drug Dev Ind Pharm 21:
19531964.
52. Nakai Y, Fukuoka E, Nakajima SI, Morita M.
1982. Physico-chemical properties of crystalline
lactose. I. Estimation of the degree of crystallinity
and the disorder parameter by an x-ray diffraction
method. Chem Pharm Bull 30:18111818.
53. Suryanarayan R. 1985. Evaluation of two concepts
of crystallinity using calcium gluceptate as a
model compound. Int J Pharm 24:117.
54. Clas SD, Faizer R, OConnor RE, Vadas EB. 1995.
Quantification of crystallinity in blends of lyophi-
lized and crystalline MK-0591using X-ray powder
diffraction. Int J Pharm 121:7379.
55. Matsuda Y, Otsuka M, Onoe M, Tatsumi E. 1992.
Amorphism and physicochemical stability of spray-
dried frusemide. J Pharm Pharmacol 44:627633.
56. Imaizumi H, Nambu N, Nagai T. 1980. Stability
and several physical properties of amorphous and
crystalline indomethacin. Chem Pharm Bull 28:
25652569.
57. Doherty C, York P, Davidson R. 1985. Factors
involved in quantitative x-ray analysis of solid
dispersions. J Pharm Pharmacol 37:57.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
58. Crocker LS, McCauley JA. 1995. Comparison of
the crystallinity of imipenem samples by X-ray
diffraction of amorphous material. J Pharm Sci 84:
226227.
59. Chen X, Bates S, Morris KR. 2001. Quantifying
amorphous content of lactose using parallel beam
X-ray powder diffraction and whole pattern fitting.
J Pharm Biomed Anal 26:6372.
60. Gao D, Rytting JH. 1997. Use of solution calori-
metry to determine the extent of crystallinity of
drugs and excipients. Int J Pharm 151:183192.
61. Saunders M, Podluii K, Shergill S, Buckton G,
Royall P. 2004. The potential of high speed DSC
(Hyper-DSC) for the detection and quantification
of small amounts of amorphous content in pre-
dominantly crystalline samples. Int J Pharm 274:
3540.
62. Hurtta M, Pitkanen I. 2004. Quantification of low
levels of amorphous content in maltitol. Thermo-
chim Acta 419:1929.
63. Ueno Y, Yonemochi E, Tozuka Y, Yamamura S,
Oguchi T, Yamamoto K. 1998 . Characterization of
amorphous ursodeoxycholic acid prepared by
spray drying. J Pharm Pharmacol 50:1213
1219.
64. Rehman M, Shekunov BY, York P, Lechuga-
Ballesteros D, Miller DP, Tan T, Colthorpe P.
2004. Optimisation of powders for pulmonary
delivery using supercritical fluid technology. Eur
J Pharm Sci 22:117.
65. Vitez IM. 2004. Utilization of DSC for pharmaceu-
tical crystal form quantitation. J Therm Anal
Calorimetry 78:3345.
66. Takahashi H, Chen R, Okamoto H, Danjo K. 2005.
Acetaminophen particle design using chitosan and
a spray-drying technique. Chem Pharm Bull 53:
3741.
67. Phillips EM. 1997. An approach to estimate the
amorphous content of pharmaceutical powders
using calorimetry with no calibration standards.
Int J Pharm 149:267271.
68. Lefort R, Gusseme AD, Willart JF, Danede F,
Descamps M. 2004. Solid state NMR and DSC
methods for quantifying the amorphous content in
solid dosage forms: An application to ball-milling
of trehalose. Int J Pharm 280:209219.
69. Kakumanu V, Bansal AK. 2002. Enthalpy relaxa-
tion studies of celecoxib amorphous mixtures.
Pharm Res 19:18731878.
70. Mooter GVD, Wuyts M, Blaton N, Busson R,
Grobet P, Augustijns P, Kinget R. 2001. Physical
stabilisation of amorphous ketoconazole in solid
dispersions with polyvinylpyrrolidone K25. Eur J
Pharm Sci 12:261269.
71. Guinot S, Leveiller F. 1999. The use of MTDSC
to assess the amorphous phase content of a
micronised drug substance. Int J Pharm 192:
6375.
DOI 10.1002/jps
 QUANTIFICATION OF AMORPHOUS/CRYSTALLINE PHASES
72. Hatta I. 1999. Conditions required for heat-
capacity measurements using modulated-tem-
perature calorimetry. Int J Pharm 192:3337.
73. Kohinata T, Fuji M, Nakamura S, Hamada N,
Yonemochi E, Terada K. 2004. Quantitative
determination of amorphous nicardipine hydro-
chloride in long acting formula (NIC-LA1) using
light anhydrous silicic acid. Chem Pharm Bull
52:14511457.
74. Briggner LE, Buckton G, Bystrom K, Darcy P.
1994. The use of isothermal microcalorimetry in
the study of changes in crystallinity induced
during the processing of powders. Int J Pharm
105:125135.
75. Chidavaenzi OC, Buckton G, Koosha F, Pathak R.
1997. The use of thermal techniques to assess the
impact of feed concentration on the amorphous
content and polymorphic forms present in spray
dried lactose. Int J Pharm 159:6774.
76. Buckton G, Darcy P, Mackellar AJ. 1995. The use
of isothermal microcalorimetry in the study of
small degrees of amorphous content of powders.
Int J Pharm 117:253256.
77. Al-Hadithi D, Buckton G, Brocchini S. 2004.
Quantification of amorphous content in mixed
systems: Amorphous trehalose with lactose. Ther-
mochim Acta 417:193199.
78. Buckton G, Darcy P, Greenleaf D, Holbrook P.
1995. The use of isothermal microcalorimetry in
the study of changes in crystallinity of spray-dried
salbutamol sulphate. Int J Pharm 116:113118.
79. Buckton G, Darcy P. 1995. The influence of
additives on the recrystallisation of amorphous
spray dried lactose. Int J Pharm 121:8187.
80. Kawakami K, Numa T, Ida Y. 2002. Assessment of
amorphous content by microcalorimetry. J Pharm
Sci 91:417423.
81. Buckton G, Darcy P. 1995. The use of gravimetric
studies to assess the degree of crystallinity of
predominantly crystalline powders. Int J Pharm
123:265271.
82. Angberg M, Nystrom C, Castensson S. 1992.
Evaluation of heat-conduction microcalorimetry
in pharmaceutical stability studies: VI. Continu-
ous monitoring of the interaction of water vapour
with powders and powder mixtures at various
relative humidity. Int J Pharm 83:1123.
83. Angberg M, Nystrom C, Castensson S. 1992.
Evaluation of heat conduction microcalorimetry
in pharmaceutical stability studies: V. A new
approach for continuous measurements in abun-
dant water vapour. Int J Pharm 81:153167.
84. Aso Y, Yoshioka S, Otsuka T, Kojima S. 1995. The
physical stability of amorphous nifedipine deter-
mined by isothermal microcalorimetry. Chem
Pharm Bull 43:300303.
85. Aso Y, Yoshioka S, Kojima S. 2001. Feasibility
of using isothermal microcalorimetry to evaluate
DOI 10.1002/jps
1663
the physical stability of amorphous nifedipine
and phenobarbital. Thermochim Acta 380:199
204.
86. Ahmed H, Buckton G, Rawlins DA. 1996. The use
of isothermal microcalorimetry in the study of
small degrees of amorphous content of a hydro-
phobic powder. Int J Pharm 130:195201.
87. Ticehurst MD, Basford PA, Dallman CI, Lukas
TM, Marshall PV, Nichols G, Smith D. 2000.
Characterisation of the influence of micronisation
on the crystallinity and physical stability of
revatropate hydrobromide. Int J Pharm 193:247
259.
88. Dilworth SE, Buckton G, Gaisford S, Ramos R.
2004. Approaches to determine the enthalpy of
crystallisation, and amorphous content, of lactose
from isothermal calorimetric data. Int J Pharm
284:8394.
89. Hogan SE, Buckton G. 2001. Water sorption/
desorption- near IR and calorimetric study of
crystalline and amorphous raffinose. Int J Pharm
227:5769.
90. Hogan SE, Buckton G. 2000. The quantification of
small degrees of disorder in lactose using solution
calorimetry. Int J Pharm 207:5764.
91. Pikal MJ, Grant DJW. 1987. A theoretical treat-
ment of changes in energy and entropy of solids
caused by additives and impurities in solid solu-
tion. Int J Pharm 39:243253.
92. Hill JO, Ojlund G, Wadso I. 1969. Thermochemical
results for tris as a test substance in solution
calorimetry. J Chem Thermodynam 1:111116.
93. Archer DG, Kirklin DR. 2000. NIST and standards
for calorimetry. Thermochim Acta 347:2130.
94. Olofsson G, Berling D, Markova N, Molund M.
2000. The dissolution of propan-1-ol and dilution of
10 wt.% propan-1-ol solution in water as calibra-
tion and test reactions in solution calorimetry.
Thermochim Acta 347:3136.
95. Wadso I. 1966. Calculation methods in reaction
calorimetry. The LKB Instru J 13:3339.
96. Willson RJ. 2002. Calorimetry. In: Haines PJ,
Haines PJ. Principles of thermal analysis and
calorimetry. Cambridge, UK: RSC Paperbacks,
pp 129165.
97. Yff BTS, Royall PG, Brown MB, Martin GP. 2004.
An investigation of calibration methods for solu-
tion calorimetry. Int J Pharm 269:361372.
98. Ward GH, Schultz RK. 1995. Process-Induced
crystallinity changes in albuterol sulphate and
its effect on powder physical stability. Pharm Res
12:773779.
99. Yonemochi E, Ueno Y, Ohmae T, Oguchi T,
Nakajima S-i, Yamamoto K. 1997. Evaluation of
amorphous ursodeoxyxholic acid by thermal meth-
ods. Pharm Res 14:798803.
100. Chadha R, Kashid N, Jain DVS. 2005. Character-
ization and quantification of amorphous content in
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
1664 SHAH, KAKUMANU, AND BANSAL
some selected parenteral cephalosporins by calori-
metric method. J Therm Anal Calorimetry 81:
277284.
101. Beezer AE. 1980. Biological microcalorimetry.
London: Academic Press.
102. Katainen E, Niemel P, Harjunen P, Suhonen J,
Jarvinen K. 2005. Evaluation of the amorphous
content of lactose by solution calorimetry and
Raman spectroscopy. Talanta 68:15.
103. Amidon GE, Bergren MS, Grant DJW, Marshall K,
Itai S. 1999. Report and recommendation of the
USP advisory panel on physical test methods:
Crystallinity determination by solution microca-
lorimetry. J Pharm Sci 25:965974.
104. Hogan SE, Buckton G. 2001. The application of
near infrared spectroscopy and dynamic vapor
sorption to quantify low amorphous contents of
cystalline lactose. Pharm Res 18:112116.
105. Mackin L, Zanon R, Park JM, Foster K, Opalenik
H, Demonte M. 2002. Quantification of low levels
(<10%) of amorphous content in micronised active
batches using dynamic vapour sorption and iso-
thermal microcalorimetry. Int J Pharm 231:227
236.
106. Mackin L, Sartnurak S, Thomas I, Moore S. 2002.
The impact of low levels of amorphous material
(<5%) on the blending characteristics of a direct
compression formulation. Int J Pharm 231:213
226.
107. Samra RM, Buckton G. 2004. The crystallisation of
a model hydrophobic drug (terfenadine) following
exposure to humidity and organic vapours. Int J
Pharm 284:5360.
108. Brittain HG. 1997. Spectral methods for the chara-
cterization of polymorphs and solvates. J Pharm
Sci 86:405412.
109. Bugay DE, Newman AW, Findlay WP. 1996.
Quantification of cefepime 2HCl dihydrate in
cefepime 2HCl monohydrate by diffuse reflectance
IR and powder X-ray diffraction techniques.
J Pharm Biomed Anal 15:4961.
110. Kamat MS, Lodder RA, DeLuca P. 1989. Near-
infrared spectroscopy determination of residual
moisture in lyophilised sucrose through intact
glass vials. Pharm Res 6:961965.
111. Martens H, Naes T. 1989. Multivariate calibra-
tion. New York: John Wiley & Sons.
112. Bai SJ, Rani M, Suryanarayan R, Carpenter JF,
Nayar R, Manning CM. 2004. Quantification of
glycine crystallinity by near infrared (NIR) spec-
troscopy. J Pharm Sci 93:24392447.
113. Gombasa A, Antal I, Szabo-Revesz P, Marton S,
Eros I. 2003. Quantitative determination of crys-
tallinity of alpha-lactose monohydrate by near
infrared spectroscopy (NIRS). Int J Pharm 256:
2532.
114. Blanco M, Villar A. 2003. Development and
validation of a method for the polymorphic analy-
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006
sis of pharmaceutical preparations using near
infrared spectroscopy. J Pharm Sci 92:823830.
115. Otsuka M, Kato F, Matsuda Y. 2000. Comparative
evaluation of the degree of indomethacin crystal-
linity by chemoinfometrical fourier-transformed
near-infrared spectroscopy and conventional pow-
der x-ray diffractometry. AAPS Pharm Sci Tech
2:18.
116. Findlay WP, Bugay DE. 1998. Utilization of four-
ier transform-Raman spectroscopy for the study of
pharmaceutical crystal forms. J Pharm Biomed
Anal 16:921930.
117. Williams AC, Cooper VB, Thomas L, Griffith LJ,
Petts CR, Booth SW. 2004. Evaluation of drug
physical form during granulation, tabletting and
storage. Int J Pharm 275:2939.
118. Taylor LS, Zografi G. 1998. The quantitative
analysis of crystallinity using FT-Raman spectro-
scopy. Pharm Res 15:755761.
119. Chan HK, Clark AR, Feeley JC, Kuo MC, Lehrman
SR, Cleland KP, Miller DP, Vehring R, Ballestros
DL. 2004. Physical stability of salmon calcitonin
spray-dried powders for inhalation. J Pharm Sci
93:792804.
120. Niemela P, Paallysaho M, Harjunen P, Koivisto M,
Lehto VP, Suhonen J, Jarvinen K. 2005. Quanti-
tative analysis of amorphous content of lactose
using CCD-Raman spectroscopy. J Pharm Biomed
Anal 37:907911.
121. Gustafsson C, Lennholm H, Iverson T, Nystrom C.
1998. Comparison of solid-state NMR and isother-
nal microcalorimetry in the assessment of the
amorphous content of lactose. Int J Pharm 174:
243252.
122. Harris RK. 1985. Quantitative aspects of high
resolution solid state nuclear magnetic resonance
spectroscopy. Analyst 110:649655.
123. Ek R, Alderborn G. 1994. Particle analysis of
microcrystalline cellulose: Differentiation between
individual particles and their agglomerates. Int J
Pharm 111:4350.
124. Ek R, Wornald P, Iversen T, Ostelius J, Nystrom
C. 1995. Crystallinity index of microcrystalline
cellulose particles compressed into tablets. Int J
Pharm 125:257264.
125. Paris M, Bizot H. 1999. Crystallinity and struc-
turing role of water in native and recrystallized
starches by 13C CP MAS NMR spectroscopy. 1:
Spectral decomposition. Carbohydr Polym 39:327
339.
126. Alamo RG, Vanderhart DL. 2000. Morphological
partitioning of ethylene defects in random propy-
lene ethylene copolymers. Macromolecules 33:
60946105.
127. Hu WG, Rohr KS. 2000. Characterization of ultra
drawn polyethylene fibers by NMR: Crystallinity,
domain sizes and a highly mobile second amor-
phous phase. Polymer 41:29792987.
DOI 10.1002/jps
 QUANTIFICATION OF AMORPHOUS/CRYSTALLINE PHASES
128. Vanderhart DL, Alamo RG. 2000. Observation of
resonances associated with stereo and region
defects in the crystalline regions of isotactic poly-
propylene: Toward a determination of morpholo-
gical partitioning. Macromolecules 33:60786093.
129. Strachan CJ, Taday PF, Newnham DA, Gordon
KC, Zeitler JA, Pepper M, Rades T. 2005. Using
terahertz pulsed spectroscopy to quantify pharma-
ceutical polymorphism and crystallinity. J Pharm
Sci 94:837846.
130. Strachan CJ, Rades T, Newnham DA, Gordon KC,
Pepper M, Taday PF. 2004. Using terahertz pulsed
spectroscopy to study crystallinity of pharmaceu-
tical materials. Chem Phys Lett 390:2024.
131. Yoshino K, Sakai T, Yamamoto Y, Inuishi Y. 1981.
Thermally stimulated current in polyacetylene.
Jpn J Appl Phys 20:867876.
132. Teyssedre G, Mezghani S, Bernes A, Lacabanne C.
1997. Thermally stimulated currents of polymers.
In: Hedvig P, Hedvig P. Dielectric spectroscopy of
polymeric materials. Bristol: Adam Hilger Ltd.,
pp 227258.
133. Bucci C, Fieschi R. 1964. Ionic thermoconductiv-
ity: Method for the investigation of polarization in
insulators. Phys Rev Lett 12:1619.
134. Huttenrauch R. 1978. Molecular galenics as the
basis of modern drug formation. Acta Pharm
Technol Suppl 6:55127.
135. Hewitt WCD, Grant DJW. 1986. True density and
thermal expansivity of pharmaceutical solids:
Comparison of methods and assessment of crystal-
linity. Int J Pharm 28:7584.
136. Price DM. 2002. Thermo-mechanical dynamic
mechanical and dielectric methods. In: Haines
DOI 10.1002/jps
1665
PJ, Haines PJ. Principles of thermal analysis and
calorimetry. Cambridge, UK: Royal Society of
Chemistry, pp 94128.
137. Jones DS. 1999. Dynamic mechanical analysis of
polymeric systems of pharmaceutical and biome-
dical significance. Int J Pharm 179:167178.
138. Royall PG, Huang C, Tang SJ, Duncan J, Van-de-
Velde G, Brown MB. 2005. The development of
DMA for the detection of amorphous content in
pharmaceutical powdered materials. Int J Pharm
301:181191.
139. Ticehurst MD, Rowe RC. 1994. Determination of
the surface properties of two batches of salbutamol
sulphate by inverse gas chromatography. Int J
Pharm 111:241249.
140. Yamamoto K, Nakano M, Arita T, Nakai Y. 1974.
Dissolution rate and bioavailability of griseofulvin
from a ground mixture with microcrystalline
cellulose. J Pharmacokinet Biopharm 2:487.
141. Sato T, Okada A, Sekiguchi K, Tsuda Y. 1981.
Difference in physico-pharmaceutical properties
between crystalline and noncrystalline 9.300 -Dia-
cetymidecamycin. Chem Pharm Bull 29:2675
2682.
142. Yamamoto K, Nakano M, Arita T, Takayama Y,
Nakai Y. 1976. Dissolution behaviour of phenytoin
from a ground mixture with microcrystalline
cellulose. J Pharm Sci 65:14841488.
143. Hasegawa S, Hamaura T, Furuyama N, Horikawa
S, Kusa A, Yonemochi E, Terada K. 2004.
Uniformity and physical states of troglitazone in
solid dispersions determined by electron probe
microanalysis and microthermal analysis. Int J
Pharm 280:3946.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 8, AUGUST 2006