Research Article: Quantification of amino acids and peptides in an ionic liquid based aqueous two-phase system by LC–MS analysis

Date Published: April 25, 2018

Publisher: Springer Berlin Heidelberg

Author(s): Sebastian Oppermann, Christina Oppermann, Miriam Böhm, Toni Kühl, Diana Imhof, Udo Kragl.


Aqueous two-phase systems (ATPS) occur by the mixture of two polymers or a polymer and an inorganic salt in water. It was shown that not only polymers but also ionic liquids in combination with inorganic cosmotrophic salts are able to build ATPS. Suitable for the formation of ionic liquid-based ATPS systems are hydrophilic water miscible ionic liquids. To understand the driving force for amino acid and peptide distribution in IL-ATPS at different pH values, the ionic liquid Ammoeng 110™ and K2HPO4 have been chosen as a test system. To quantify the concentration of amino acids and peptides in the different phases, liquid chromatography and mass spectrometry (LC–MS) technologies were used. Therefore the peptides and amino acids have been processed with EZ:faast™-Kit from Phenomenex for an easy and reliable quantification method even in complex sample matrices. Partitioning is a surface-dependent phenomenon, investigations were focused on surface-related amino acid respectively peptide properties such as charge and hydrophobicity. Only a very low dependence between the amino acids or peptides hydrophobicity and the partition coefficient was found. Nevertheless, the presented results show that electrostatic respectively ionic interactions between the ionic liquid and the amino acids or peptides have a strong impact on their partitioning behavior.

Partial Text

During the past few decades ionic liquids have become an essential part in chemical and biochemical research (Wasserscheid and Welton 2008). Ionic liquids are organic salts with unique physical properties, for example high thermal stability, low viscosity and a negligible vapor pressure. Below 100 °C, ionic liquids exist in a liquid state (Seddon 1997). These properties depend on their structure. Steric effects and charge delocalization of their ions hinder the formation of a stable crystal lattice (Kyte and Dolittle 1982). Already low thermal energy is sufficient to overcome the lattice energy and to break up the solid crystalline structure (Kyte and Dolittle 1982). Ethyl ammonium nitrate was the first known ionic liquid and was described by Walden in 1914 (Walden 1914). A number of various ionic liquids have been described in the last decades. Some examples of water miscible ionic liquids are shown in Table 1 (Wasserscheid and Welton 2008).Table 1Examples for ionic liquids

The investigated amino acids have different structural requirements and influences to the partitioning behavior: leucine as an amino acid with a long aliphatic chain, glycine as the smallest natural amino acid, serine which has an alcohol group at its side chain, phenylalanine with a phenyl group, glutamic acid with a second carboxyl group and finally histidine which has an imidazole group (Table 2). The results show that there has to be a interaction between the cation of Ammoeng 110™ (AE110, IL) and the negatively charged carboxyl groups of the amino acids (Fig. 2). Because of this phenylalanine (Phe), glutamic acid (Glu) and histidine (His) are enriched in the ionic liquid containing upper phase of the ATPS at both pH values.




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