Transcript AnionsCE
Concentration Determination
via Leucigenin Quenching
and Indirect CE
Christopher
Hampton,
Dr. Eamonn F.
Healy,
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Chemistry
Department,
St. Edward’s
University,
Austin TX 78704
Background on Capillary
Electrophoresis
Capillary (20-200x10^-6 M inner diameter)
High voltages
Electroosmotic Flow
Electrophoretic Flow
UV Spectroscopy
Benefits
Minute samples (uL Samples may be used)
Fast separation
Linear quantization
Electrophoretic/osmotic Flow
Electrophoretic Flow
µ=[Ld/tm]/[V/Lt]
Ld = length to detector
tm = migration time
V = voltage
Lt = total length
Electroosmotic Flow
Veo=[Edc(Zp)/4πn]/E
Edc = dielectric constant
Zp = Zeta potential
n = viscosity
E = electric field strength
CE Experimental Method
Instrument Used
P/ACE 5000 CE
Fused silica capillaries at
57cm x 75µm
Working length 50cm
6 second pressure
injections
Chemicals Used
All of ACS reagent grade
All solutions were made
with Millipore water
Experimental Method, contd.
BGE Composition
Chromic Acid
Hydroxybenzenesulfonic Acid
5 mM
5 mM
Ratio of BGE’s 1:1
Buffered with diethanolamine to pKa of
DEA
pH 9.2
Indirect Detection
Background Electrolytes
(BGE)
BGE1, BGE2
UV absorbing species
Analytes displace
electrolytes
Results in “negative”
peak, analytes do not
absorb
Negative peak flipped
by software
Indirect Detection Methods
BGE composition
Chromic Acid
CAS [7738-94-5]
5mM
Hydroxybenzenesulfonic Acid
CAS [1333-39-7]
5mM
Ratio 1:1
DEA (Diethylanolamine)
CAS [111-42-2]
pKa of DEA
~pH 9.2
Chromate Chromatograph,
5mM
OH-BSA Chromatograph,
5mM
Chloride Chromatograph,
0.2mM
Fluorescence Work
Lucigenin has been
widely known to be
quenched by Chloride
anions for a long time
This phenomenon
has not been
extensively studied,
and no detailed
analytical
characterization has
been done.
Fluoroscopy
Benefits
Small samples (3mL)
Widely available
Limitations
Not all materials
fluoresce
Cost of fluorescent
materials
Detection limits
Fluoroscopy Experimental
Method
Instrument Used
Shimadzu RF 5301 PC Fluorometer
Suprasil 4mL Fluorescence cuvette (1cm
base)
Chemicals Used
All of ACS reagent grade
Chloride solutions were made from a volumetric
NaCl standard solution obtained from Sigma
Aldrich (1g Cl- / 100g water)
All solutions were made with Millipore water
Experimental Method, contd.
Standards prepared
Lucigenin
concentration from an
ethanol stock, diluted
in water
Solutions were
combined in a capped
cuvette, and vortexed
for 30-45 seconds
Experimental Method, contd.
An excitation
spectrum was
obtained at 505nm
Maximum peak
intensity and
differentiation was
consistently observed
at 368 and 432 nm
0.1 µM Lucigenin Quenching
0.1 µM Lucigenin Regression
0.05 µM Lucigenin Quenching
0.05 µM Lucigenin Regression
Preliminary Results and
Questions
50 nano-molar
concentration of
Lucigenin, and a 50
micro-molar Clsolutions.
1000:1 ratio of Cl- to
Lucigenin.
This can be further
reduced, but with an
increased of noise to
signal ratio loss
Still
working at
concentrations that are
showing very distinct
patterns
We are approaching
the limits of detection
of our instrument and
operator
Background
Contamination of our
water?
Discussion
Haddad and Dobble1 showed that a mixture of
CrO4 and OHBSA could be used to separate a wide
array of of cations and anions.
•
Applying the methods used, it can be shown that
with few modifications, one can successfully
separate mixtures of cations and anions in
concentrations in the parts-per-billion (ppb) ranges.
•
This work focused solely on chloride (Cl-), even at
the low concentrations optimum peak shape and
consistent retention times were achieved.
•
Ok, but is it real?
Still working at
concentrations that
are showing very
distinct patterns
We are approaching
the limits of detection
of our instrument and
operator
Contamination of our
water?
Where are we going from
here?
CE is going to make or
break it.
Buffer has been
problematic
Repeat of 25 nm
fluorescence data set
(clean it up some)
Lower the Cl- : Lucigenin
ratio to 1:100 (I.e. 101µM Cl-)
Determine Cl specificity
vs. other halide salts
Acknowledgements
We gratefully acknowledge the support of the
Welch Foundation in the form of a Departmental
Research Grant
Dr. Eamonn Healy and the Saint Edward’s
University chemistry faculty
Mr. Jon Steuernegel, Nathan Svadlenak, Brent
Polishak, et al, for their continued assistance
and support