Transcript AnionsCE

Concentration Determination
via Leucigenin Quenching
and Indirect CE
Christopher
Hampton,
Dr. Eamonn F.
Healy,
QuickTime™ and a
Photo - JPEG decompressor
are needed to see this picture.
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