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