APPLICATION NOTE – Scanning Electron Microscopy
Introduction
Researchers continually strive to develop
pharmaceutical remedies with the intent of combating
different disease states, alleviating suffering, and
thwarting negative health effects. While different
pharmaceutical formulations can be developed to target
a unique set of symptoms, they all share one
fundamental principle: improve a patient’s quality of life
by way of therapeutic effectiveness. Formulation
scientists are therefore tasked with leveraging the
properties of drugs to ensure treatments counteract a
patient’s symptoms quickly and vigorously.
Enhancing therapeutic effectiveness
At the core of therapeutic effectiveness is
bioavailability, which describes the degree to which a
compound integrates into the systemic circulation and
becomes available at the site of drug action. It is a
property related to the dosage form such as solution,
suspension, tablet, capsule, powder, and elixir [1]. The
bioavailability of orally ingested formulations is affected
by aqueous solubility, drug permeability, dissolution
rate, first-pass metabolism, pre-systemic metabolism,
and susceptibility to efflux mechanisms [2].
Nanoparticles and drug delivery
Once administered into the body, the efficacy of
drugs is influenced by several barriers existing between
the point of entry and the site of therapeutic action. The
use of nanoparticles as a drug delivery mechanism is
advantageous for optimizing bioavailability because
smaller particles more efficiently traverse circulatory
streams with improved absorption and increased
solubility. The versatility of nanoparticles for drug
delivery is the subject of investigation, especially for
optimizing bioavailability. Nanoparticles may have the
active pharmaceutical ingredient (API) either chemically
attached, encapsulated, adsorbed, or entrapped. Figure
1 illustrates the advantages of nanoparticles for drug
delivery. The substantial surface-to-volume ratios
provided by the small size of the nanoparticles are at the
core of what makes nanoparticles an ideal drug delivery
1
vehicle. However, the size, composition, and porosity
influence the efficacy of drug release which makes the
characterization and analysis of these morphological
parameters vital.
Characterizing nanoparticles
Figure 1: Key advantages of using nanoparticles for drug
delivery.
Particle size and distribution, the level of
agglomeration, porosity, and surface morphology are
among the most important characteristics of
nanoparticles that require analysis for pharmaceutical
applications. These parameters govern, in vivo
distribution, toxicity, targeting abilities, and drug loading
and release abilities. The agglomeration state of
nanoparticles may influence the transport and cellular
internalization as well.
Nanoparticle fabrication methods commonly
result in broad particle size distributions, aggregated
clumps, and foreign particulates. A quantitative
understanding of these attributes is, therefore,
necessary to monitor particle fabrication methods and
ensure that the resulting combination of APIs and
excipients (inactive pharmaceutical ingredients) lead to
predictable and stable dosages.
Dynamic light scattering (DLS) is an important
tool to characterize the size of nanoparticles. DLS is
based on the random diffusive Brownian motion of
particles in solution wherein larger particles move slower
22-SEM01
APPLICATION NOTE – Scanning Electron Microscopy
and scatter more light than smaller particles. The
hydrodynamic size of the particles can be calculated from
their speed. However, DLS cannot differentiate between
large particles and agglomerates of smaller particles. The
ability to identify nanoparticle agglomeration is crucial
for understanding the apparent surface area of a
particular formulation as well as potential issues with
uneven API distribution.
Mass spectrometry methods are often used to
gather chemical composition information of
nanoparticles. But these methods are rather
rudimentary when it comes to determining the size or
size distribution of the nanoparticle or correlating the
chemical composition with a location.
When considering the shortcomings of these
methods, it becomes clear that a more advanced
technique is needed to survey the size distribution,
morphology, and chemical composition of nanoparticles
efficiently.
SEM for characterizing nanoparticles
Scanning electron microscopy (SEM), by it being
a direct visualization tool, is ideal for the characterization
and analysis of particle size and morphological
parameters. Supplementing SEM with energy-dispersive
X-ray spectroscopy (EDS) enables the identification and
quantification of the chemical composition of
nanoparticles with high spatial resolution. This capability
can be invaluable when studying the loading efficiency of
API on nanoparticles and identifying foreign particulates.
Desktop SEM
The last couple of decades have seen an increase
in the popularity of desktop SEMs over floor model SEMs.
Floor model SEMs are larger and require a specialized
room to operate effectively, whereas the desktop SEMs
can be set up on a standard laboratory bench.
Technological innovations have driven considerable
improvement in the resolution of desktop SEMs to below
10 nm. Their lower purchase and operation cost,
combined with their compact design, makes them an
2
ideal tool for integrating into existing pharmaceutical
nanoparticle formulation workflows.
Desktop SEM is a user-friendly and cost-effective
solution to characterize nanoparticles. Their compact
nature means that analysis can be performed in the same
space where the nanoparticles are fabricated thus
cutting down lead times for generating relevant data.
Formulation scientists can use desktop SEM to detect
quality characteristics and potential defects on the fly,
thus streamlining the R&D process.
A few examples demonstrating how desktop
SEM can be used to characterize nanoparticles are
described below. Each example highlights the various
capabilities of Phenom desktop SEMs while emphasizing
the convenience of having powerful SEM
instrumentation in-house.
Figure 2: Microparticles of polyvinylpyrrolidone imaged by a
Phenom desktop SEM. The image shows agglomeration and
morphological details.
I. Examine particle morphology: To reveal the
wide range of morphological details, from larger
agglomerates to smaller features like satellites, high-
resolution images with good signal-to-noise ratios are
required. A Phenom XL desktop SEM equipped with a
cerium hexaboride crystal (CeB6) electron source was
used to image polyvinylpyrrolidone particles. The image
in Figure 2 reveals the morphology of the particles and
the surface features stand out.
22-SEM01
APPLICATION NOTE – Scanning Electron Microscopy

II. Imaging non-conductive samples: Since many of

the nanoparticles used in pharmaceuticals are non-
conductive, charges tend to build up on them and reduce
the quality of images. Sputter-coating a thin layer of
platinum or gold onto the sample can help dissipate
charging and improve image quality. However, sputter
coating may introduce artifacts and can alter the surface
morphology of certain samples. Having low-vacuum and
low-kV capabilities is desirable for SEM analysis when it
comes to imaging non-conductive samples. Running an
SEM in low vacuum mode can negate the need for
sputter coating entirely due to the presence of ionized
gas that acts to neutralize surface charges.

Additionally, low-kV imaging results in less charging due

to the shallower penetration depth of incident electrons.
A Phenom Pharos desktop SEM, equipped with a field
emission gun source (FEG) enables image acquisition at
low-kV and low-vacuum conditions. Figure 3 shows an
example of images collected in a Phenom Pharos at low
kV, comparing API in nasal spray suspension before and
after spraying.
III. Analyze nanoparticle size distribution:
Nanoparticle fabrication methods tend to result in a wide
range of diameters and by extension – a wide range of
surface areas. As such, it is critical to ensure that batches
of nanoparticles are consistent with each other and
remain within an acceptable range to efficiently absorb
into the systemic circulation. Both drug diffusion and
degradation of the microsphere matrix are tightly linked
to average diameter, so it is essential to obtain accurate
size distribution statistics for quality control.
One module of Phenom SEM software – ParticleMetric –
automates the particle analysis process while providing
rich summary statistics for a wide variety of particle
properties. The software analyzes thousands of particles
acquired from SEM image sets and yields data tables,
histograms, and customized reports based on user
applications. Figure 4 shows an example of the live
Figure 3: Images of nasal spray collected on a desktop Phenom
Pharos under low kV and low vacuum conditions. Circled in yellow,
the API proceeded through the spraying process without
experiencing any changes to surface area/size. Each image is at a
different magnification, but the consistency in particle size can be
quickly confirmed.
interactive display of the Phenom ParticleMetric
software.
IV. Analyze API distribution: Since APIs tend to be
portioned at the microgram scale (or even smaller), a
significant mass fraction of drug formulation consists of
excipients to produce dosage forms of a reasonable,
ingestible size. Therefore, it is critical to understand the
relationship between the two components – essentially
how they adsorb and intermingle. The images in Figure 5
convey nitrogen and phosphorous API adsorption onto
excipient materials: the left image is an SEM image, and

3
22-SEM01
APPLICATION NOTE – Scanning Electron Microscopy

Figure 4: Automatic particle analysis dashboard showing summary statistics of 15 different particle properties (e.g., area, circle equivalent
diameter, perimeter, circumscribed circle diameter, convex hull perimeter, intensity mean, major axis, minor axis, etc). Acquired with a Phenom
desktop SEM.

Figure 5: An API comprised of mostly nitrogen is identified to concentrate heavily in concave areas of the carbon, hydrogen, and oxygen-based
excipient. Acquired with a Phenom desktop SEM and analyzed with integrated EDS (energy dispersive spectroscopy) features.

4
22-SEM01
APPLICATION NOTE – Scanning Electron Microscopy

the right image is a corresponding EDS map showing the
elemental distribution and confirming higher
concentrations of API in concave areas of the excipient.
Both the image and EDS map were acquired using a
Phenom desktop SEM.
V. Microparticle characterization: Microspheres
are often used to achieve controlled release of drugs to
targeted sites. A few critical factors for this application
include the diffusion of the drug from the microparticle
and the degradation of the microspheres themselves.
Figure 6 presents data from microspheres of varying
pore densities. The density of pores observed can be
correlated with the release rate of drugs from these
spheres. The shape, size, and morphology of the spheres
affect their performance. Since sputter coating of the
microspheres can potentially obscure the pore
morphology, these microspheres were imaged in low kV
and low vacuum conditions using a Phenom XL desktop
SEM.
thoroughly understood to best leverage the nanoscale
properties for targeted and therapeutic effectiveness,
quality control, and manufacturability. Accessing
properties in domains smaller than those reachable by
optical light demands the use of electron microscopy.
Monitoring the right nanoparticle attributes can be time-
consuming and challenging without the aid of adept
tools.
The Phenom desktop SEMs include several features that
make them ideal for the analysis of nanoparticles. These
include automated high-throughput image acquisition,
automated particle size analysis, integrated EDS with
mapping, long-lifetime CeB6/FEG sources, low-kV, and
low-vacuum mode, all in a compact table-top design.
References
[1] Allam, Ahmed. (2011). Bioavailability: A Pharmaceutical
Review. J Novel Drug Deliv Tech. 1.
[2] Jia L. Nanoparticle Formulation Increases Oral
Bioavailability of Poorly Soluble Drugs: Approaches
Experimental Evidences and Theory. Curr Nanosci. 2005 Nov
1;1(3):237-243.

[3] K.T. Savjani, A.K. Gajjar, J.K. Savjani. Drug solubility:

importance and enhancement techniques. ISRN Pharm
(2012), pp. 1-10, 10.5402/2012/195727

Nanoscience Instruments, Inc.

Figure 6: Porosity of microspheres. Phenom XL images of 480-758-5400
microspheres with different pore densities. If the surface info@nanoscience.com
contains too many micropores (left image), the initial www.nanoscience.com
release of active pharmaceutical agent will be too high.
Whereas when the surface is smooth (right image), drug
release will be more gradual.

Conclusion
The core aspect of pharmaceutical product
development is an investigation into the nanoscale
behavior of the drugs’ constituents. Physical and
chemical characteristics of the API and excipient must be

5
22-SEM01