# New Method for Simultaneous Arsenic and Selenium Speciation Analysis in Seafood and Onion Samples
## 3 Materials and Methods
### Method Development for Simultaneous Speciation Analysis  
Analytical Column and Mobile Phase Effect  
Based on the type of analysis and separate arsenic, antimony, chromium, and selenium forms, different analytical columns were tested. The main element for this research was arsenic, so the analysis was optimized in all conditions to separate the As(III), As(V), DMA, MMA, and AsB forms. After finding the optimal conditions and separating all of the arsenic forms, further elements were added, and the effectiveness of the analysis was checked. Out of the various types of columns, the following were tested:  
- PRP-X100 (250 mm \(\times\) 4.1 mm, 10 \(\upmu\)m), packing material type: PS-DVB/Trimethyl ammonium exchanger (Hamilton, Reno, NV, USA);  
- PRPX-200 PEEK (250 mm \(\times\) 4.6 mm, 10 \(\upmu\)m), packing material type: PSDVB/Sulfonic Acid (Hamilton, USA);  
- BioWAX (50 mm \(\times\) 2.1 mm, 5 \(\upmu\)m), packing material type: nonporous/dietyloamine (Agilent, Santa Clara, CA, USA);  
- Supelco SAX (250 mm \(\times\) 4.6 mm, 5 \(\upmu\)m), packing material type: silica gel, spherical particle platform/propyltrimethylammonium phase (Sigma-Aldrich, USA);  
- Waters SAX (250 mm \(\times\) 4.6 mm, 5 \(\upmu\)m), packing material type: silica-based quaternary ammonium bonded sorbent (Waters, Milford, MA, USA);Dionex CS5A (250 mm \(\times\) 4 mm, 9 \(\upmu\)m), packing material type: latex, DVB/Cation: Sulfonic Acid/Anion: Quaternary Ammonium (Thermo, Waltham, MA, USA);  
- Dionex CG5A (50 mm \(\times\) 4 mm, 9 \(\upmu\)m), packing material type: latex, DVB/Cation: Sulfonic Acid/Anion: Quaternary Ammonium (Thermo, USA);  
- Dionex AS22 (250 mm \(\times\) 4 mm, 6 \(\upmu\)m), packing material type: DVB/Alkanol Quaternary Ammonium Ion (Thermo, USA);  
- Dionex AG22 (250 mm \(\times\) 4 mm, 6 \(\upmu\)m), packing material type: DVB/Alkanol Quaternary Ammonium Ion (Thermo, USA).  
Based on the literature describing arsenic, antimony, chromium, and selenium speciation analysis, different mobile phases were chosen for the experiment. Of the various types of mobile phases, ammonium carbonate (NH4)2 CO3, ammonium bicarbonate NH4 HCO3, nitric acid HNO3, ammonium nitrate NH4 NO3, and ethylenediamine tetraacetic acid EDTA were examined (Sigma-Aldrich, USA). The best resolution results were obtained for NH4 NO3. The addition of methanol was also tested. Three concentrations of methanol (1%, 2%, and 3%) in the mobile phase were tested. The pH value was modified in a range from 3.0 to 9.0. It was possible to obtain most of particular forms of analyzed metals with a good resolution only with the application of the Dionex IonPac AS22 with the Dionex IonPac CG5A guard column. Moreover, the most satisfactory analytical signals were obtained for concentrations of 1% and 2% methanol, so for further analysis 1% methanol was chosen. Unfortunately, these conditions were not optimal for speciation analysis of all forms of the chosen elements. A particular problem was the pH of the mobile phase, which was the key to the selection of analytes for further analysis. None of the forms of chromium could be separated in any analytical combination. The antimony forms were separated only in an acidic pH. However, it was possible to separate all arsenic and selenium species, though this required the use of an alkaline pH. In connection with the above, it was decided to continue and further optimize the analysis of only the arsenic and selenium species. The best resolution of the selenium forms was obtained in an acidic pH of 3.0, but at such an acidic pH we could not obtain a signal from arsenobetaine, which required a pH of 9.0. It was this pH that was chosen for further analysis. The influence of the column type and mobile phase on the separation effect is presented in table (Table 2).