Preliminary study of two steps aqueous two-phase extraction combined with high performance liquid chromatography for saliva protein selective separation

Saliva protein separation by one-step aqueous two-phase extraction (ATPE) in the system of 15% (mass ratio if not specified) PEG-4000/8% NaH₂PO₄ was presented. The proteins extracted from the top and bottom phases were separated and characterized by high performance liquid chromatography (HPLC) respectively. Under the optimized gradient elution procedure, more saliva protein peaks in the two phases were shown. Saliva protein peaks in 50 min gradient were divided into ten specific groups which were classified into six sections based on the protein distribution in the top and bottom phases. Then, the top or bottom phase was performed the second step ATPE. Possible reasons for protein partition and retention difference are discussed briefly. Results show that some proteins can be separated completely in one-step ATPE, while some can be separated in the following two-step ATPE. The combination of two steps ATPE and HPLC provides a new way to achieve selective separation.