Ordered
alphabetically by student's last name
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Adelman |
Bourreza | Dignan | Knerr | Meyers | Passarelli | Reed | Schnitker | Wagner |
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| Bazzoli | Carter |
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Koyoshi |
Nuzzio |
Pirnot |
Rhoades |
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Zony | |
| Benavidez |
Currier | Hullmann |
Lansing | Pagels | Tereniak |
Zucker |
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Growth Preference between Laminin and Fibronectin Patterning
Peter Adelman, Soonmoon Yoo, Bill Theilacker, Jeffery Twiss, and Thomas P. Beebe Department of Chemistry and Biochemistry and the Following an injury,
neurons in the peripheral nervous
system (PNS) are capable of re-extending neurites or axons and
restoring
function. The central nervous system (CNS) only shows regeneration
under very
special circumstances. If the mechanism
for this regenerative growth could be understood, then it might be
possible to
facilitate PNS regeneration and greatly improve outcomes for CNS
regeneration
(e.g., spinal cord injury). In large
part, axon growth is guided by extracellular matrix (ECM)
macromolecules, which
can encourage or inhibit neurite growth. Here,
I am setting the groundwork to ask how axons
distinguish between
common ECM components found in the PNS and CNS. We have devised a means
to
determine how neurons show preference between the two supportive
matrices,
laminin and fibronectin. To accomplish this, we micro-contact printed
two
dimensional growth substrates with the laminin and fibronectin proteins
in adjacent
stripes. These substrates were analyzed
for surface chemistry and then for neurite growth promotion. Earlier studies from our lab had shown that
axons of rat sensory neurons prefer laminin to fibronectin, though
recent
literature suggests neurite outgrowth may favor the protein-protein
interaction
between laminin and fibronectin, rather than having a preference for
one of the
two (Hodgkinson, et al., 2007). To more
closely address this issue of axonal substrate preference, dissociated
adult
rat dorsal root ganglia were plated onto engineered coverslips with 40
µm wide
stripes of fibronectin and laminin. These
cultures consist of a mixture of sensory neurons and
the
non-neuronal 'Schwann' cells. The number of axon/neurite crossing from
one
substrate to the other was quantitated and correlated with Schwann cell
contact. Supported by HHMI. |
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Identifying Biomarkers of the MHC Gene to Investigate Chemosensory Discrimination Tyler C. Bazzoli and Steven D. Brown Department of Chemistry and Biochemistry Variability in the highly polymorphic genes of the major histocompatibility complex (MHC) plays a fundamental role in influencing the odor of urinary volatiles in the common house mouse (Mus musculus). To better understand the mechanisms underlying MHC-dependent chemosensory signals, it is critical to characterize the chemical nature of MHC-dependent odorants. Urine samples from MHC-genotyped mice were analyzed using capillary gas chromatography. By employing multivariate analysis and pattern recognition techniques on the chromatographic data, it is possible to classify mice by their MHC genotype and to explore which chemical compounds are biomarkers capable of discrimination. Soft Independent Modeling of Class Analogy (SIMCA) of the data was performed and indicated that the observed genotypes were easily distinguishable and well separated, a strong indication that specific compounds differentiated the genotypes. Based on the SIMCA model, a graph of the discriminating power of each compound within the model was generated. The compounds that exhibited a high level of genotype-discriminating power were dimethyldisulfide, methyl methylsulfenylmethyl disulfide, Z-5,5-Dimethyl-2-ethylidenetetrahydrofuran, and E-5,5-Dimethyl-2-ethylidenetetrahydrofuran.The structural similarity between the two disulfide compounds along with the similarity of the two tetrahydrofuran compounds suggest that there may be a biochemical explanation for the role that these four compounds play in differentiating mice with dissimilar MHC genes. The ability to qualitatively determine specific biomarkers associated with genetic differences has an invaluable application in the field of genomic medicine. Further exploration of the relationship between chemistry and genetics promises new treatments for gene-linked ailments. Supported by the Howard Hughes Medical Institute. |
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Evaluating Anti-Androgen Resistance by In-Vitro Selection
Aly Bourreza and John Koh Department of Chemistry and Biochemistry Anti-androgens,
such as flutamide and bicalutamide (Casodex®), used alone or in
conjunction
with chemical castration, have been used in the treatment of prostate
cancer (PCa)
for decades. However, as many as 30%- 40%
of patients treated with anti-androgens acquire a therapy-resistant
phenotype after
one to three years of treatment. Furthermore,
some patients experience clinical improvement upon cessation of
anti-androgens,
a condition known as anti-androgen withdrawal syndrome.
Anti-androgen withdrawal syndrome has been
associated with mutations to the AR that cause an agonist response to
anti-androgens and is one of the most difficult forms of PCa to treat. The
goal of this project is to redesign anti-androgens to evade molecular
mechanisms that lead to anti-androgen withdrawal syndrome in androgen
independent
PCa. To accomplish this, novel analogs,
PLM1 and PLM6, were developed that remain antagonists towards three AR
mutants
associated with anti-androgen withdrawal syndrome.
In this study, long-term growth analyses of
the human LNCaP cell line in the presence of PLM1 and PLM6 versus
bicalutamide
have been performed to identify potential AR mutants that would
represent an
anti-androgen withdrawal phenotype. After
approximately 4- 6 weeks, resistant colonies developed and were
selected. DNA sequence data of the
resistant colonies
show mutations to the AR. Current
studies are underway to examine the effects of drug withdrawal and
changing the
drug treatment. This work is supported
by the Howard Hughes Medical Institute Undergraduate Science Education
program
and the National Institutes of Health, NIDDK; 3-R01-DK054257-09.
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Development of a Fluorescent Sensor of ErbB2 Kinase Activity for Application in Breast Cancer Research Ann Benavidez and Neal Zondlo Department of Chemistry and Biochemistry When
breast cancer develops in an individual, ErbB2 kinase signaling becomes
deregulated. The resultant uncontrolled phosphorylation leads to
functional
changes in multiple proteins, altering cellular behavior. To explore
ErbB2
kinase signaling, we sought to develop fluorescent sensors of ErbB2
activity. Using
solid-phase peptide synthesis, chemical phosphorylation, and reverse
purification techniques, two tyrosine kinase-inducible domains were
created
which contain ErbB2 recognition sites and have varying electrostatics
at the
N-terminus. The sequences are based off of a EF hand motif, modified so
as to respond
to phosphorylation. Since peptides bind terbium ions and fluoresce when
phosphorylated and are unstructured when nonphosphorylated, I
hypothesized that
the phosphorylated peptides would bind Tb3+ well and display
great
terbium luminescence, while the nonphosphorylated peptides would poorly
bind Tb3+
and display correspondingly weaker terbium luminescence. Accordingly, I
have
sought to purify phosphorylated and nonphosphorylated versions of the
peptides
to investigate this phenomenon. In the future, phosphorylation behavior
upon
exposure to ErbB2 will be examined and the necessary modifications will
be performed
in order to build a tyrosine kinase-inducible domain with optimal
specificity
for ErbB2. (Funded by Chemistry Alumni Scholars Program).
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A Study
of the Effect of a t-Butyl Group on the α-Carbon
of Chloroformate Esters 1Department of Chemistry, 2Department of Chemistry & Biochemistry, Northern |
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Jenna M. Currier,
Peychii Lee, and Roberta
F. Colman Adenylosuccinate lyase (ASL) is an enzyme that catalyzes two distinct reactions in the de novo synthesis of purine nucleotides. The metabolic importance of this enzyme is shown by the observation of autism, mental retardation, developmental delay, epilepsy and muscle wasting in patients with ASL deficiency. ASL deficiency is caused by single point mutations of the gene encoding ASL, with many patients being heterozygous for two different point mutations. We now seek to determine whether human ASL subunits with different point mutations can form hybrids to restore enzymatic activity. The disease-associated mutations R194C and R396C were constructed by site-directed mutagenesis, expressed in E. coli and purified. R396C has a specific activity only 27% that of WT human enzyme, but is comparable to WT enzyme in stability; while R194C has similar specific activity to WT, but is more thermally unstable than is the normal enzyme. Each enzyme has a 6-histidine tag at the N-terminus, allowing it to bind reversibly to a nickel-NTA column. To separate hybrids as a result of complementation, the His-tag from R396C was cleaved with thrombin, rendering it unable to bind to the Ni-NTA column. Complementation experiments were conducted by brief exposure of an enzyme mixture (R194C + R396C) to 1.5M guanidine HCl followed by a five-fold dilution, causing dissociation then reassociation of the 4 subunits. Using an imidazole gradient on a Ni-NTA column, the hybrid enzymes were isolated, as confirmed through N-terminal sequencing. Kinetic and stability measurements are now being performed. (Supp. by HHMI.)
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Stabilization of Human Adenylosuccinate Lyase
Through
Site-Directed Mutagenesis of Cysteine Residues Adenylosuccinate
lyase (ASL) is a metabolically important enzyme that catalyzes two
distinct
reactions in the purine nucleotide biosynthesis pathway: conversion of
adenylosuccinic acid to adenosine monophosphate (AMP) and fumarate, and
conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to
aminoimidazole carboxamide ribotide (AICAR) and fumarate. Human ASL
exhibits
marked instability compared to the ASL of other species because it is
easily
oxidized and is cold-inactivated. It contains 13 cysteine residues that
are not
conserved among other species and that are not involved at the
catalytic site.
Seven of these residues are close enough to form unwanted disulfide
bonds upon
oxidation: Cys266, Cys304, Cys305, Cys98,
Cys99, Cys172, and Cys173. It is
possible to
stabilize human ASL by keeping it in the presence of reducing agents
like
dithiothreitol (DTT), but this is cumbersome and often impractical. In
order to
stabilize the enzyme, alanine residues were systematically substituted
for
cysteine through site-directed mutagenesis and overexpressed in E. coli. The Vmax and Km
for Adenylosuccinate were obtained for the WT, C266/304/305A, C98/99A,
C266/304/305/172A, and C266/304/305/173A. All of these enzymes had
comparable
values for both the Km and Vmax. Circular
dichroism was
used to compare the secondary structure of the enzymes, and the
activity of the
WT and mutants were tested under oxidative stress at 37ºC with
hydrogen
peroxide and oxidized glutathione. (Funding provided by HHMI.) |
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Synthesis and Characterization of New Alkaline-Earth Metal Indium-Antimonides Jonathan Hullmann and Svilen Bobev Department of Chemistry and Biochemistry Thermoelectric
materials may provide a unique solution to the continual problem of
increased
energy demand due to their ability to convert thermal energy into
electricity,
and vise versa. These materials can
be
utilized in refrigeration units without the need for environmentally
harmful
CFC’s. Another promising application of
these materials is the conversion of low-level waste heat from
automobile
engines to electricity; decreasing the use of fossil fuels. However, thermoelectric materials do not
have widespread application today because they are inefficient. In 2006, the intermetallic compound Yb14MnSb11
was synthesized and found to have four times greater
efficiency than the
thermoelectric material currently used by NASA, SiGe.
This discovery prompted renewed interest in
structurally related antimonides.
Intrigued, we began investigating similar compounds. Until now we have synthesized and
structurally characterized three new materials: Eu11InSb9,
Yb11InSb9 and Sr11InSb9,
the
properties of which are currently being studied. During these
investigations,
several other new compounds were also discovered: Ba4Sb3,
Sr4Sb3 and Eu4Sb3. These three compounds cannot be made using
the traditional solid-state approaches, suggesting In inclusions or
substitutions in the crystal structure.
In addition, another “first-of-a-kind” compound, Ba4In3Sb4,
was recently discovered. Exploration of
the synthetic conditions for Ba4In3Sb4 versus
the still unknown Ba14InSb11 is ongoing. This research was funded by the Howard Hughes
Medical Institute.
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Metal-triggered Hydrogelation
of
Designed β-hairpin Peptides Patrick J. Knerr,
Christopher Micklitsch, Colin
Thorpe, and Joel P.
Schneider
Department of Chemistry and Biochemistry Peptides have been designed which undergo an environmentally-triggered folding transition from random coil to a β-hairpin conformation, responsive to changes in temperature, pH and ionic strength. These folded, amphiphilic β-hairpins subsequently self-assemble to form a crosslinked, fibrillar network, yield a self-supporting, rigid hydrogel. This work reports a new folding trigger: zinc-induced hydrogelation. An unnatural, negatively-charged α-amino acid with a strong propensity to chelate zinc was synthesized and incorporated into the hydrophobic face of a β-hairpin sequence composed of two strand regions connected by a four residue type II’ β-turn. The negative charge of this residue interferes with hydrophobic interactions necessary for folding and self-assembly; hydrogelation can only be accomplished when a zinc ion chelates to the residue and neutralizes charge to allow adoption of the amphiphilic β-hairpin. With further sequence modulation, a peptide was designed which can fold and self-assemble at physiological pH only when a stoichiometric amount of the zinc trigger is added. Such a responsive design is of particular interest in sensor technologies to detect toxic levels of zinc in the environment, in bioremediation and in the design of microfluidic devices and novel nanomaterials. Funding has been supplied by the National Science Foundation and the Arnold and Mabel Beckman Foundation. |
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Synthesis of 1,1’- Diaminoferrocene James Lansing, John Young, Wes Monillas, David Grieco, and Klaus Theopold Department of Chemistry and Biochemistry 1,1’-Diaminoferrocene
(DAFc) is a
redox active ligand whose derivatives have been employed in several
uses, such
as an olefin polymerization catalyst.[1]
Following an improved literature procedure,
its synthesis has become significantly easier and made it accessible as
a
ligand.[2] As of now, its
uses when complexed to a cobalt cation have yet to be explored. We hope to complex it to a cobalt cation and
oxidize the resulting coordination compound, with the hopes of
preparing cobalt
in a high oxidation state and with the potential for Proton Coupled
Electron
Transfer. The
first step in the synthesis of
DAFc involved a TMEDA/ n-butyl lithium complex to form
1,1’-dilithioferrocene(1). It
was seen that the addition of even small amounts of air
cause
immediate decomposition of the lithiated product, thus employing
Schlenk line
techniques and inert gas protection were of utmost importance. After workup was performed in a nitrogen
atmosphere, 1 was reacted with
1,1,2,2-tetrabromoethane
to brominates the species, forming 1,1’-dibromoferrocene(2). Both temperature and
rate of addition of tetrabromoethane played a role in the reaction,
which
optimized to a 70% yield. Once 2
was formed, it was reacted with
cuprous chloride in ethanol and aqueous sodium azide.
Following the formation of 1,1’-diazidoferrocene(3), a hydrogenation with a palladium on
carbon catalyst should yield the diamino product. As
of yet, our efforts to produce and isolate
the azide and subsequent amino derivative have failed, but further
exploration
is being undertaken. Supported
by Chemistry Alumni Undergraduate
Research Fellowship Program
[1]Arnold, J., Shafir, A. Ferrocene-Based Olefin Polymerization Catalysts: Activation, Structure, and Intermediates. Organometallics 2003, 22, 567-575 [2]Arnold, John, et al. Synthesis, Structure, and Process of 1,1’-Diamino- and 1,1’-Diazidoferrocene, Organometallics 2000, 19, 3978-3982 |
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Isolation and Purification of Vanadium Haloperoxidase Mutants for Characterization by 51V Solid-State NMR Department of Chemistry & Biochemistry Vanadium haloperoxidases
are a specific class of vanadium containing enzymes commonly found in
marine
algae, lichens and terrestrial fungi. These enzymes are the most
efficient
halide oxidants known to date. Vanadium containing compounds have shown
excellent potential in the treatment of diabetes, particularly as
insulin
enhancing compounds, as well as in the treatment of some forms of
cancer.
However, in order for these compounds to be useful in biomedical
applications,
the structure of the vanadium active sites and the mechanism of their
biochemical activity need to be determined. The focus of our work is to
understand the catalytic mechanism of vanadium haloperoxidases and
their active
site mutants by utilizing 51V solid-state NMR. This
knowledge is
expected to be important in the design of artificial vanadium enzymes
with
tuned halogenating activities. We have established protocols for the
expression, isolation and purification of recombinant vanadium
chloroperoxidase
(VCPO) using a Saccharomyces cerevisiae overexpression
system (Strain BJ1991 with the vector PTNT14).
We have successfully isolated and purified two VCPO active site
mutants: R360A
and R490A. We have quantified the concentration of the purified protein
and
begun to crystallize the wild type protein using the hanging drop
method to
form well-ordered crystals. These protocols are necessary for
preparation of
the protein samples for subsequent 51V solid-state NMR
spectroscopy
studies. 51V solid-state NMR will be used to directly probe
the
diamagnetic “spectroscopically” silent vanadium sites in biological
systems,
which will encompass both insulin mimetics and vanadium
haloperoxidases. Funding
for this research was provided through the Chemistry & Biochemistry
Alumni
Scholars Program.
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Effects of sorbitol on folding/unfolding and aggregation of a-chymotrypsinogen Rebecca K. Pagels, Rebecca K. Brummitt, and Christopher J. Roberts Department of Chemical Engineering Irreversible,
non-native aggregation is a constant concern for the biopharmaceutical
industry
throughout the production process and storage as it can result in loss
of
viable product. a-Chymotrypsinogen (aCgn) is
a well-studied model protein for non-native aggregation under acidic
solution
conditions where the aggregates remain soluble. The focus of this study
is to
assess the impact of a canonical stabilizing additive (sorbitol) on
different
stages of aggregation. The primary methods of experimentation were
differential
scanning calorimetry, size exclusion high performance liquid
chromatography,
circular dichroism, and fluorescence spectroscopy. These techniches
helped determine
the change in Gibbs free energy of unfolding (DGunf
), monomer loss kinetics, secondary structure, and
tertiary structure, respectively, of the protein with increasing
sorbitol concentration
at fixed temperature. Changes in DGunf with increasing sorbitol concentrations were
qualitatively consistent with observations in the literature in that
addition
of sorbitol (slightly) stabilized the native monomer state over the
unfolded
state. Addition of sorbitol caused only minor changes to the rate of
aggregation, with the exception of a notable increase in aggregation
rate at
intermediate sorbitol concentration. This anomaly cannot be explained
by
changes in the thermodynamics of unfolding, and the data is currently
inconclusive concerning changes in the secondary and tertiary structure
of the
unfolded monomer. Future work will involve light scattering and seeding
experiments in an attempt to separate the intrinsic aggregate
nucleation and
growth timescales.
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Preparation and Pasteur Resolution of Racemic Hydrobenzoin Michael T. Pirnot and Douglass F. Taber Department of Chemistry and Biochemistry In 1849, Louis Pasteur separated racemic tartrate through crystallization and physical separation of the enantiomerically-pure crystals. This has come to be called “Pasteur resolution.” It was also reported many years ago that racemic hydrobenzoin also resolved spontaneously in crystallization. Thinking that this would make a good organic teaching lab experiment, we were faced with two questions: 1. How to prepare racemic hydrobenzoin; and 2. How to carry out the crystallization so the enantiomeric crystals could be separated. |
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Darneisha Reed1,
Fumie Koyoshi, Malcolm
J.
D’Souza1, Dennis
N. Kevill2
1Department of Chemistry, 2Department of Chemistry & Biochemistry, Northern |
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Tara Rhoades and John
Koh <>
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Stephen J. Tereniak, Matthew T.
Kieber-Emmons, Nathan A.
Eckert, and Charles G.
Riordan (1) Ragsdale, S. W. Crit. Rev. Biochem. Mol. Bio. 2004, 39, 165. (2) Eckert, N. A.; Dougherty, W. G.; Yap, G. P. A.; Riordan, C. G. J. Am. Chem. Soc. 2007, 129, 9286. |
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Synthesis and Applications of Complex Molecules with a-Substituted Guanidiniums Sasha J. Wagner and Neal J. Zondlo Department of Chemistry and Biochemistry The guanidinium
functional group enables and mediates
interactions between proteins, DNA, and RNA. Therefore,
guanidiniums have a variety of applications in
molecular
recognition and pharmaceutical chemistry. To
encourage specific recognition, an amino acid is
coupled to a chiral
base molecule and the amino acid is guanylated. This
process converts the free amine to a guanidinium. Specific
activity of the previously-described
molecule can be modified by manipulating the identity and
stereochemistry of
the base molecule, such as a 1,2-diaminocyclohexane, and the side chain
and
stereochemistry of the amino acid. The
characteristics of the diamine and the properties of the amino acid
side chain
allow different parts of the overall molecule to work in cooperation
with one
another to increase its affinity for specific target areas. We have isolated both R,R and
S,S-(1,2)-diaminocyclohexane and coupled them to Boc-protected
L-phenylalanine. After Boc deprotection,
the free amines were guanylated using Moroder's reagent.
It was imperative that the Boc-protected
guanidinium groups were deprotected under mildly acidic conditions, in
order to
minimize epimerization. These complex
molecules, with -substituted guanidiniums, can be used for specific
recognition of target areas, such as RNA or telomeric G-quadruplexes,
whose
elongation is involved in the majority of cancers. Supported by the
Science and
Engineering Scholars Program.
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Chati L. Zony, Anastasia.G. Fuzaylova and Roberta F. Colman Department of Chemistry and Biochemistry Glutathione
S-Transferase pi (GST pi) plays a vital role in cell detoxification and
has
recently been shown to interact with c-Jun N-terminal kinases (JNKs), a
kinase family
involved in the Mitogen Activated Protein Kinase cascade. Normally,
JNKs are
essential components involved in the initiation of apoptosis triggered
during
periods of cellular stress. In cancerous cells, GST pi is overexpressed
and has
been postulated to inactivate JNKs through the formation of a complex,
which in
turn inhibits apoptosis, leading to tumor survival and proliferation.
The
complex has been observed in vivo,
but isolation and characterization of this complex in vitro
has not been accomplished. We now seek to
isolate the complex of GST pi
and two isoforms of JNK : JNK1a2 and JNK2a2. Current work with JNK2a2
involves
its incubation with a histidine-tagged GST pi in HEPES buffer (pH 7.8),
containing
12.5% 1,6-hexanediol to dissociate the dimers, followed by dialysis in
the same
buffer but lacking 1,6-hexanediol, thus allowing complex formation. The
complex
is purified using a nickel- nitriloacetic acid agarose column and an
imidazole
gradient. Preliminary results show that under current conditions, the
complex is
formed but dissociates during the imidazole gradient. Studies are
currently
underway to determine more favorable conditions to stabilize and purify
the
complex. We have also inserted the cDNA for JNK1a2 into pET15b, a
bacterial
vector which yields the target protein with an N-terminal
histidine-tag. This
new construct will be used to express and purify JNK1a2 for similar
experiments
in complex isolation. By understanding the basis for the JNK/GST pi
interaction, inhibitors of the interaction may be designed which should
enhance
the beneficial effects of apoptosis to tumor tissues. Funded by the
Ronald E.
McNair Scholars Program and the Howard Hughes Medical Institute. |
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Aerosol Analysis Using A Scanning Mobility Particle Sizer Steven M. Zucker, Julie A. Lloyd, Murray V. Department of Chemistry and Biochemistry The goal of
atmospheric aerosol research is to understand the source,
transformation and
fate of airborne particles. Laboratory generated particles play
an
important role in this work, serving both as well-characterized
reactants in
laboratory studies of gas-particle reactions and as test particles for
evaluating and checking the performance of instrumentation. The goal of this project is to generate and
characterize test particles for a new instrument that analyzes organic
and
biological components in particles. A
Scanning Mobility Particle Sizer (SMPS) was used to measure the number
and mass
concentrations of erythromycin particles. Solutions
were prepared at various concentrations and all
particles were
generated with a constant output atomizer. It
was observed that as the concentration of solute
increases, the mass
concentration of particles increases linearly, but the number of
particles
increases logarithmically. The effect of
solvent on particle generation was also studied. The
number of erythromycin particles was
found to be the same whether using 100% methanol or 100% ethanol. When the erythromycin solute was 50% water by
volume, the number of particles generated decreased significantly. The above results were obtained with a
constant output atomizer. Current and
future
work will focus on the use of other aerosol generation methods such as
the
electrospray ionization generator and the Collison Nebulizer. Funding for this research was provided by the
Delaware Space Grant Consortium. |