# P S A NALYTICAL

P S ANALYTICAL

UK: Arthur House, Crayfields Industrial Esta te, Main Road, Orpington, Kent BR5 3HP, UK USA: 1761 W. Hillsboro Blvd., Suite 318, Deerfield Beach, FL 33442, USA Tel: +1 (954) 429 1577 Fax: (954) 429 1601 E-mail: usa@psanalytical.com www.psanalytical.com 

# Application Note 147

SELENIUM SPECIATION IN WASTEWATER BY HPLC-HG-AFS

Selenium typically exists in oxic surface waters in the form of two major species: the selenite (SeO 3 2- ; oxidation state +IV) and selenate (SeO4 2- ;oxidation state +VI). Thermodynamically, Selenite is thermodyna mically instable and would convert into selenate over time, but the kinetic of this reaction is so slow that it does not occur in
ambient waters, unless microbes are invo lved. Nonetheless, well-oxidized waters tend to contain mostly selenite. This application note describes a simple and robust method to separate and quantify the two selenium species, using HPLC with post column oxidation coupled to hydride generation atomic fluorescence s pectrometry (HPLC-HG-AFS).



### Procedure The system involves the coupling of PSA speciation 10.820 (HPLC, 6-port valve, UV cracker, heater and cooler in one module) to the PSA Millennium Excalibur. SAMS+ software is used to control the autosampler, the speciation module 10.820 and the

Millennium Excalibur for data collecti on. The method settings used for this application are shown in the software window in figure 1.

Figure 1: Method Settings of the Millennium Excalibur System

Figure 2: Schematic Diagram of the HPLC-HG-AFS System for the Speciation of SeleniumP S ANALYTICAL

UK: Arthur House, Crayfields Industrial Esta te, Main Road, Orpington, Kent BR5 3HP, UK Tel: +44 (0) 1689 891 211 Fax: +44 (0) 1689 896009 E-mail: psa@psanalytical.com
USA: 1761 W. Hillsboro Blvd., Suite 318, Deerfield Beach, FL 33442, USA Tel: +1 (954) 429 1577 Fax: (954) 429 1601 E-mail: usa@psanalytical.com www.psanalytical.com Figure 2 shows a schematic di agram of the system. 250 μL sample solution are injected onto a HPLC column, where an isocratic program is used to separate the species. As the species elute from the column they mix with a stream of KBr/HCl and pass through a heater at 120 °C, a UV cracker and a cooler. The solvent stream is then mixed with NaBH 4 forming volatile hydrides which are swept from the gas liquid separator in a stream of argon. This stream flows through a Perma Pure TM membrane to remove any moisture and then into the detector. Here a hydrogen diffusion flame, fuelled by the excess hydrogen produced in the hydride generation step, converts the hydrides to free ground state selenium atoms. These atoms are then excited by a selenium boos ted discharge hollow cathode lamp, and the occurring fluorescence is detected by a photomultiplier tube. Separation and detection of Se(IV) and Se(VI) takes place in less than 14 minutes. The chromatographic and chemical conditions are outlined in table 1. Species in 3 waste water samples were identified on the basis of their retention times. 4 samples were spiked and the spike-recovery calculated. Precision for the all species is below 1% RSD, and limits of detection for Se(IV) and Se(VI) are 0.05 and 0.05 μg L -1 respectively, as shown in Table 2.

Table 1: Instrument Settings of the HPLC-HG-AFS System for Selenium Speciation

HPLC Conditions Column Hamilton PRP-X 100 (250 X 4,6 mm) 10 μm
Injected volume 250 μ l Mobile phase flow rate 1 mL min - 1

Chemical Conditions
Mobile phase 5 mM Citric Acid, 2% Methanol, pH 5.9
Acid 6 M HCl, 5% m/v KBr, 2 ml min - 1

Reductant 0.7% m/v NaBH 4 in 0.1M NaOH, 4.5 ml min - 1

Table 2: Figure of Merit for Selenium Speciation Using Millennium Excalibur

Se(IV) Se(VI) Slope 165.75 164.34

Calibration correlation coefficient 1.0000 0.9998 SD baseline (300 counts) 0.208 0.214 MDL 3 (μg/L) 0.05 0.06 MQL 10 (μg/L) 0.17 0.2

Figure 3: Calibration curves for Se(IV) and Se(VI).P S ANALYTICAL

UK: Arthur House, Crayfields Industrial Esta te, Main Road, Orpington, Kent BR5 3HP, UK Tel: +44 (0) 1689 891 211 Fax: +44 (0) 1689 896009 E-mail: psa@psanalytical.com
USA: 1761 W. Hillsboro Blvd., Suite 318, Deerfield Beach, FL 33442, USA Tel: +1 (954) 429 1577 Fax: (954) 429 1601 E-mail: usa@psanalytical.com www.psanalytical.com 

### Figure 4: (a) Chromatograms for standards of Se(IV) and Se(VI): Blank, 1, 5, 10, 20, and 40 μg L -1



### . (b) overlayed chromatograms for waste water 3 (unspiked and spiked with 10 μg/L) and 10 μg/L Se(IV) and Se(VI) standard

Table 3: Figure of Merit for Selenium Speciation Using Millennium Excalibur

Sample Se(IV) Spiked Se(IV)

R% Se(VI) Spiked Se(VI) R% Dilution factor

1 <0.5 89% 0.03 ± 0.01 90% 1

2 <0.5 88% 215.5 ± 8.5 89% 20

3 <0.5 88% 2.1 ± 0.0 92% 1

The 3 samples contained only Se(VI), the Se(IV) concentra tion was below the limit of

detection. The concentration of Se(VI) varied between 0. 03 and 215.5 μg/L. Spike recoveries (10 μg/L Se(IV) and Se(VI) were added) was in the range of 88-89 % for Se(IV) and 89-92 % for Se(VI).

Conclusion One of the main advantages of atomic fluorescence is the sensitivity, stability and linear range it offers. Additionally, HG-AFS is a reliable and inexpensive method of carrying out selenium speciation. The separation of the two common selenium species in water (Se(IV) and Se(VI)) can be carried out very rapidly within 14 min.

For further information on this method and required extractions, please contact P S Analytical.