P S ANALYTICAL

P S ANALYTICAL 

UK: Arthur House, Crayfields Industrial Estate, 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 (SeO3

2- ; oxidation state +IV) and selenate (SeO42-;oxidation state +VI). 

Thermodynamically, Selenite is thermodynamically 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 involved. 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 spectrometry (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 collection. 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 Selenium
P S ANALYTICAL 

UK: Arthur House, Crayfields Industrial Estate, 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 diagram 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 NaBH4 forming 

volatile hydrides which are swept from the gas liquid separator in a stream of argon. This 

stream flows through a Perma PureTM 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 boosted 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 μl 

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 NaBH4 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 Estate, 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) concentration 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.