[Study on the combination of isotachophoresis and high performance liquid chromatography for the separation of...

A method on the combination of isotachophoresis (ITP) and high performance liquid chromatography (HPLC) for the separation and analysis of the complex samples containing proteins and metals is proposed. The instrumental system consisted of a self-made Model OCEP-1 isotachophoresis analyser, main parts of Waters high performance liquid chromatograph and a self-made Model IHI-1 interface. The interface is simple and convenient to use. The injection volume of the interface was determined accurately with a neutralization titration method. In ITP experiment the leading electrolyte was 2.5 mmol/L HCl containing 30 mmol/L of 18-crown-6; the terminating electrolyte was 5 mmol/L Tris+Cl-(pH 6.0); ITP current, 40 microA; detection of metal ions, potential gradient detector. In HPLC experiment the column was a mu-Bondapak C18, reversed-phase column, 4 mm x 200 mm, 10 microns, 12.5 nm; the mobile phase A, V(isopropanol with 5% volume fraction):V(butanol) = 2:1 containing 2.2 mmol/L HCl (pH 2.63); the mobile phase B, V(isopropanol with 50% volume fraction):V(butanol) = 2:1; low pressure gradient elution, mobile phase B changed from 0 to 70% in 17.5 minutes; flow rate, 1.0 mL/min; detection wavelength 225 nm; temperature, 25 degrees C. The experimental results showed that proteins were interfered in the determination of the metals with ITP method. It also showed that HPLC with the use of C18 column can separate the proteins but not the metal ions. By using this new combination system, a complex sample of the proteins and metal ions was injected to the HPLC, the proteins Lys, BSA and OVA were well separated in HPLC, while NH4+, K+, Na+, and Ca2+ were less retained and eluted first. Then the latter was introduced into the system of the ITP with the aid of the interface for separation. Satisfactory results were obtained.