HPLC Separation of Underivatized Amino Acids in Buffer-Less Mode on Coresep 100 Core-Shell Mixed-Mode Column

Application description

Amino acids are very polar compounds which are used as a building blocks in pharmaceutical industry. They are widely used as supplements and food additives. Depending on the pH of the mobile phase they can exist in acidic, basic or zwitter-ionic form. At pH below 3, amino acids are basic in nature and have the highest hydrophobicity. Within pH of 3 to 7 amino acids are in zwitter-ionic form where they are the most hydrophilic. There is no mechanism of retention on reversed-phase column and amino acids are not retained. Bufferless ion-separation (BLIS) was introduced by SIELC as a way to retain and analyze amino acids in reversed-phase cation-exchange mode. This mode allows to analyze amino acids as zwitter-ions without any ions/buffers in the mobile phase. The method was adopted for analysis of amino acids on core-shell mixed-mode columns. A similar approach on a reversed-phase core-shell column results in distorted peaks and no separation or significant retention to achieve baseline separation.

Conditions of Experiment
Column: Coresep 100
Separation Modes: reversed-phase and cation-exchange
Column Dimenstions: 3.2 x 50 mm, 2.7 um, 90A
Mobile Phase: 20% ACN with no additives
Detection: UV 205 nm, ELSD
Sample: 0.3 mg/ml
Injection: 1 uL
Flow rate: 1 ml/min
Class of compounds: Amino acid, Supplement
Nature of compounds: Hydrophilic, Polar, Zwitterionic
Compounds: Methionine, Tyrosine, Phenylalanine