Aquious Halide Concentration Determination via Leucigenin Quenching and Indirect CE Christopher Hampton, Dr. Eamonn F. Healy, Chemistry Department, St. Edward’s University, Austin TX 78704

Background on Capillary Electrophoresis Capillary (20-200x10^-6 M inner diameter) Capillary (20-200x10^-6 M inner diameter) High voltages High voltages Electroosmotic Flow Electroosmotic Flow Electrophoretic Flow Electrophoretic Flow UV Spectroscopy UV Spectroscopy Benefits Benefits Minute samples (uL Samples may be used) Minute samples (uL Samples may be used) Fast separation Fast separation Linear quantization Linear quantization

Electrophoretic/osmotic Flow Electrophoretic Flow Electrophoretic Flow µ=[Ld/tm]/[V/Lt] µ=[Ld/tm]/[V/Lt] Ld = length to detector Ld = length to detector tm = migration time tm = migration time V = voltage V = voltage Lt = total length Lt = total length Electroosmotic Flow Electroosmotic Flow Veo=[Edc(Zp)/4πn]/E Veo=[Edc(Zp)/4πn]/E Edc = dielectric constant Edc = dielectric constant Zp = Zeta potential Zp = Zeta potential n = viscosity n = viscosity E = electric field strength E = electric field strength

CE Experimental Method Instrument Used Instrument Used P/ACE 5000 CE P/ACE 5000 CE Fused silica capillaries at 57cm x 75µm Fused silica capillaries at 57cm x 75µm Working length 50cm Working length 50cm 6 second pressure injections 6 second pressure injections Chemicals Used Chemicals Used All of ACS reagent grade All of ACS reagent grade All solutions were made with Millipore water All solutions were made with Millipore water

Experimental Method, contd. BGE Composition BGE Composition Chromic Acid Chromic Acid 5 mM 5 mM Hydroxybenzenesulfonic Acid Hydroxybenzenesulfonic Acid 5 mM 5 mM Ratio of BGE’s 1:1 Ratio of BGE’s 1:1 Buffered with diethanolamine to pKa of DEA Buffered with diethanolamine to pKa of DEA pH 9.2 pH 9.2

Indirect Detection Background Electrolytes (BGE) BGE 1, BGE 2 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 [ ] 5mM Hydroxybenzenesulfonic Acid CAS [ ] 5mM Ratio 1:1 DEA (Diethylanolamine) CAS [ ] 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 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. This phenomenon has not been extensively studied, and no detailed analytical characterization has been done.

Fluoroscopy Benefits Benefits Small samples (3mL) Small samples (3mL) Widely available Widely available Limitations Limitations Not all materials fluoresce Not all materials fluoresce Cost of fluorescent materials Cost of fluorescent materials Detection limits Detection limits

Fluoroscopy Experimental Method Instrument Used Instrument Used Shimadzu RF 5301 PC Fluorometer Shimadzu RF 5301 PC Fluorometer Suprasil 4mL Fluorescence cuvette (1cm base) Suprasil 4mL Fluorescence cuvette (1cm base) Chemicals Used Chemicals Used All of ACS reagent grade All of ACS reagent grade Chloride solutions were made from a volumetric NaCl standard solution obtained from Sigma Aldrich (1g Cl- / 100g water) 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 All solutions were made with Millipore water

Experimental Method, contd. Standards prepared Standards prepared Lucigenin concentration from an ethanol stock, diluted in water Lucigenin concentration from an ethanol stock, diluted in water Solutions were combined in a capped cuvette, and vortexed for seconds Solutions were combined in a capped cuvette, and vortexed for seconds

Experimental Method, contd. An excitation spectrum was obtained at 505nm An excitation spectrum was obtained at 505nm Maximum peak intensity and differentiation was consistently observed at 368 and 432 nm 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 Cl- solutions. 50 nano-molar concentration of Lucigenin, and a 50 micro-molar Cl- solutions. 1000:1 ratio of Cl- to Lucigenin. 1000:1 ratio of Cl- to Lucigenin. This can be further reduced, but with an increased of noise to signal ratio loss 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 Still working at concentrations that are showing very distinct patterns We are approaching the limits of detection of our instrument and operator We are approaching the limits of detection of our instrument and operator Background Contamination of our water? Background Contamination of our water?

Discussion Haddad and Dobble 1 showed that a mixture of CrO 4 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 Still working at concentrations that are showing very distinct patterns We are approaching the limits of detection of our instrument and operator We are approaching the limits of detection of our instrument and operator Contamination of our water? Contamination of our water?

Where are we going from here? CE is going to make or break it. CE is going to make or break it. Buffer has been problematic Buffer has been problematic Repeat of 25 nm fluorescence data set (clean it up some) Repeat of 25 nm fluorescence data set (clean it up some) Lower the Cl- : Lucigenin ratio to 1:100 (I.e µM Cl-) Lower the Cl- : Lucigenin ratio to 1:100 (I.e µM Cl-) Determine Cl specificity vs. other halide salts 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 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 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 Mr. Jon Steuernegel, Nathan Svadlenak, Brent Polishak, et al, for their continued assistance and support