1 Capillary Electrophoretic Determination of Selenium and Tellurium Oxyanions in Bacterial Cultures by Bala Krishna Pathem and Thomas G. Chasteen Department of Chemistry Sam Houston State University

2 Instrument configuration Source: ceandcec.com

3 Flow reversal For Anions: For Anions: –In order to speed up the separation, the EOF can be reversed by addition of cationic surfactants. –A negative voltage is applied. –We have used TTAB and HTAB in our work.

4 Source: ceandcec.com

5 Method development We developed a method for the identification of selenium species in presence of TSB or LB bacterial growth medium, complex and well-defined media. We developed a method for the identification of selenium species in presence of TSB or LB bacterial growth medium, complex and well-defined media. We applied the method for simultaneous estimation of selenate and selenite or tellurite in live cultures during their biological reduction by bacteria. We applied the method for simultaneous estimation of selenate and selenite or tellurite in live cultures during their biological reduction by bacteria.

6 Capillary Fused silica capillaries were used for all the studies done in this research. Fused silica capillaries were used for all the studies done in this research. Dimensions: Dimensions: 50 cm effective length, 75 µ m i.d, 375 µ m O.D.

7 Method Buffer: 15 mM KH 2 PO mM TTAB Buffer: 15 mM KH 2 PO mM TTAB pH pH Injection Pressure: 0.5 psi Time: 5 sec Injection Pressure: 0.5 psi Time: 5 sec Separation Voltage: – 25.0 KV Separation Voltage: – 25.0 KV Capillary temp: 25 o C Capillary temp: 25 o C Run time: 5 min Run time: 5 min Detection: 190 nm for selenium oxyanions Detection: 190 nm for selenium oxyanions 220 nm for tellurium oxyanions 220 nm for tellurium oxyanions

8 Bacterial growth conditions Two bacterial species were used in our research. Two bacterial species were used in our research. 1.Precultures of Bacillus sp. were grown in TSB (pH 7.0) at 30 o C and then amended with 1.0 mM selenate. 2. Precultures of genetically modified clone of E.coli K- 12 (1VH) were grown in LB medium with Ampicillin (pH 7.0) at 37 o C and then amended with 0.05 mM tellurite.

9 Figure 1

10 Bioreduction of selenate by Bacillus sp. Figure 2

11 Figure 3

12 Figure 4

13 Figure 5

14 Bioreduction of 0.05 mM tellurite by 1VH Figure 6 0 h 5 h 24 h after inoculation mAU TeO 3 2-

15 Figure 7

16 Conclusions Once bacteria enters stationary phase (4 h): Once bacteria enters stationary phase (4 h): –Rate of selenate reduction decreases –Selenite reduction process begins or increases significantly, 25% drop in SeO 3 2- in 1 h Varied bacterial response to toxic salts Varied bacterial response to toxic salts –Selenite is more toxic than selenate for both these organisms, based on specific growth rates

17 Conclusions Method offers excellent LOD (3S/N) Method offers excellent LOD (3S/N) –Selenate : 1.0 ppm or mM –Selenite : 0.25 ppm or mM Linearity Linearity –Selenate: (0.1 mM to 1.0 mM) –Selenite : (0.1 mM to 1.0 mM)

18 Conclusions This method affords high sample through- put and minimal or no sample preparation for biological samples. This method affords high sample through- put and minimal or no sample preparation for biological samples. Perfect tool for the Qualitative or Quantitative analysis of selenium and tellurium oxyanions in solution. Perfect tool for the Qualitative or Quantitative analysis of selenium and tellurium oxyanions in solution. Applicable for analysis in environmental or agricultural samples. Applicable for analysis in environmental or agricultural samples.

19 Acknowledgements Sam Houston State University’s Faculty Enhancement Research Fund The Robert A. Welch Foundation This work was supported by

20 THANK YOU QUESTIONS??