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Aquious Halide Aquious Halide Concentration Concentration
Determination via Determination via Leucigenin Quenching Leucigenin Quenching
and Indirect CEand Indirect CEChristopher Hampton,
Dr. Eamonn F. Healy,
Chemistry Department,
St. Edward’s University,
Austin TX 78704
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Background on Capillary Background on Capillary ElectrophoresisElectrophoresis
Capillary (20-200x10^-6 M inner Capillary (20-200x10^-6 M inner diameter)diameter)
High voltagesHigh voltages Electroosmotic FlowElectroosmotic Flow Electrophoretic FlowElectrophoretic Flow
UV SpectroscopyUV Spectroscopy BenefitsBenefits
Minute samples (uL Samples may be used)Minute samples (uL Samples may be used) Fast separationFast separation Linear quantizationLinear quantization
Electrophoretic/osmotic Electrophoretic/osmotic FlowFlow
Electrophoretic FlowElectrophoretic Flow µ=[Ld/tm]/[V/Lt]µ=[Ld/tm]/[V/Lt] Ld = length to detectorLd = length to detector tm = migration timetm = migration time V = voltageV = voltage Lt = total lengthLt = total length
Electroosmotic FlowElectroosmotic Flow Veo=[Edc(Zp)/4πn]/EVeo=[Edc(Zp)/4πn]/E Edc = dielectric Edc = dielectric
constantconstant Zp = Zeta potentialZp = Zeta potential n = viscosityn = viscosity E = electric field E = electric field
strengthstrength
CE Experimental MethodCE Experimental Method Instrument UsedInstrument Used
P/ACE 5000 CEP/ACE 5000 CE Fused silica capillaries Fused silica capillaries
at 57cm x 75µmat 57cm x 75µm Working length 50cmWorking length 50cm
6 second pressure 6 second pressure injectionsinjections
Chemicals UsedChemicals Used All of ACS reagent All of ACS reagent
gradegrade All solutions were made All solutions were made
with Millipore waterwith Millipore water
Experimental Method, Experimental Method, contd.contd.
BGE CompositionBGE Composition Chromic AcidChromic Acid
5 mM5 mM Hydroxybenzenesulfonic AcidHydroxybenzenesulfonic Acid
5 mM5 mM Ratio of BGE’s 1:1Ratio of BGE’s 1:1 Buffered with diethanolamine to pKa Buffered with diethanolamine to pKa
of DEAof DEA pH 9.2pH 9.2
Indirect DetectionIndirect Detection
Background Electrolytes (BGE)
BGE1, BGE2
UV absorbing speciesAnalytes displace electrolytes
Results in “negative” peak, analytes do not absorbNegative peak flipped by software
Indirect Detection Indirect Detection MethodsMethods
BGE compositionChromic Acid
CAS [7738-94-5] 5mM
Hydroxybenzenesulfonic Acid
CAS [1333-39-7]5mM
Ratio 1:1DEA (Diethylanolamine)
CAS [111-42-2] pKa of DEA~pH 9.2
Chromate Chromate Chromatograph, 5mMChromatograph, 5mM
OH-BSA Chromatograph, OH-BSA Chromatograph, 5mM5mM
Chloride Chromatograph, Chloride Chromatograph, 0.2mM0.2mM
Fluorescence WorkFluorescence Work Lucigenin has been Lucigenin has been
widely known to be widely known to be quenched by quenched by Chloride anions for Chloride anions for a long timea long time
This phenomenon This phenomenon has not been has not been extensively studied, extensively studied, and no detailed and no detailed analytical analytical characterization characterization has been done.has been done.
FluoroscopyFluoroscopy
BenefitsBenefits Small samples Small samples
(3mL)(3mL) Widely availableWidely available
LimitationsLimitations Not all materials Not all materials
fluorescefluoresce Cost of fluorescent Cost of fluorescent
materialsmaterials Detection limitsDetection limits
Fluoroscopy Fluoroscopy Experimental MethodExperimental Method
Instrument UsedInstrument Used Shimadzu RF 5301 PC FluorometerShimadzu RF 5301 PC Fluorometer Suprasil 4mL Fluorescence cuvette (1cm Suprasil 4mL Fluorescence cuvette (1cm
base)base) Chemicals UsedChemicals Used
All of ACS reagent gradeAll of ACS reagent grade Chloride solutions were made from a volumetric Chloride solutions were made from a volumetric
NaCl standard solution obtained from Sigma NaCl standard solution obtained from Sigma Aldrich (1g Cl- / 100g water)Aldrich (1g Cl- / 100g water)
All solutions were made with Millipore waterAll solutions were made with Millipore water
Experimental Method, Experimental Method, contd.contd.
Standards preparedStandards prepared Lucigenin Lucigenin
concentration from concentration from an ethanol stock, an ethanol stock, diluted in waterdiluted in water
Solutions were Solutions were combined in a combined in a capped cuvette, capped cuvette, and vortexed for and vortexed for 30-45 seconds30-45 seconds
Experimental Method, Experimental Method, contd.contd.
An excitation An excitation spectrum was spectrum was obtained at 505nmobtained at 505nm
Maximum peak Maximum peak intensity and intensity and differentiation was differentiation was consistently consistently observed at 368 observed at 368 and 432 nmand 432 nm
0.1 µM Lucigenin 0.1 µM Lucigenin QuenchingQuenching
0.1 µM Lucigenin 0.1 µM Lucigenin RegressionRegression
0.05 µM Lucigenin 0.05 µM Lucigenin QuenchingQuenching
0.05 µM Lucigenin 0.05 µM Lucigenin RegressionRegression
Preliminary Results and Preliminary Results and QuestionsQuestions
50 nano-molar 50 nano-molar concentration of concentration of Lucigenin, and a 50 Lucigenin, and a 50 micro-molar Cl- micro-molar Cl- solutions.solutions.
1000:1 ratio of Cl- 1000:1 ratio of Cl- to Lucigenin.to Lucigenin.
This can be further This can be further reduced, but with reduced, but with an increased of an increased of noise to signal ratio noise to signal ratio lossloss
Still working at Still working at concentrations that concentrations that are showing very are showing very distinct patternsdistinct patternsWe are We are approaching the approaching the limits of detection limits of detection of our instrument of our instrument and operatorand operatorBackground Background Contamination of Contamination of our water?our water?
DiscussionDiscussion
•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 Ok, but is it realreal?? Still working at Still working at
concentrations that concentrations that are showing very are showing very distinct patternsdistinct patterns
We are We are approaching the approaching the limits of detection limits of detection of our instrument of our instrument and operatorand operator
Contamination of Contamination of our water?our water?
Where are we going from Where are we going from here?here?
CE is going to make or CE is going to make or break it.break it.
Buffer has been Buffer has been problematicproblematic
Repeat of 25 nm Repeat of 25 nm fluorescence data set fluorescence data set (clean it up some)(clean it up some)
Lower the Cl- : Lower the Cl- : Lucigenin ratio to Lucigenin ratio to 1:100 (I.e. 10-1µM Cl-)1:100 (I.e. 10-1µM Cl-)
Determine Cl Determine Cl specificity vs. other specificity vs. other halide saltshalide salts
AcknowledgementsAcknowledgements
We gratefully acknowledge the support of We gratefully acknowledge the support of the Welch Foundation in the form of a the Welch Foundation in the form of a Departmental Research GrantDepartmental Research Grant
Dr. Eamonn Healy and the Saint Edward’s Dr. Eamonn Healy and the Saint Edward’s University chemistry faculty University chemistry faculty
Mr. Jon Steuernegel, Nathan Svadlenak, Mr. Jon Steuernegel, Nathan Svadlenak, Brent Polishak, et al, for their continued Brent Polishak, et al, for their continued assistance and supportassistance and support