Glycine

HPLC Analysis of Sugars, Amino Acids and Carboxylic Acids on Amaze TH Mixed-Mode Column
Application description

Mixed-mode stationary phases were designed to retain and separate compounds with drastic difference in properties using one column and method. Carboxylic acids, amino acids and sugars are poorly retained in reversed-phase chromatography. Derivatization procedures, ion-pairing reagents and HILIC columns can be used tp analyze these complex mixtures. The use of ion-pairing reagent makes MS detection problematic due to volatility issues of IP and signal suppression in MS. HILIC mixed-mode columns, like Amaze TH, have polar ionizable groups on the surface which help significantly increase retention time of polar ionizable analytes like amines, amino acids, carboxylic acids and sugars. We have developed a method for separation and retention of succinic acid, phenylalanine, sucrose, glycine, aspartic acid and raffinose. Isocratic conditions provide separation within 15 minutes with good peak shape and retention control. Retention time on Amaze TH column is controlled by amount of ACN, buffer concentration, buffer ph and buffer nature. Method can be used for other amines, amino acids, carboxylic acids and sugars where fast MS-compatible separation is required.

Conditions of Experiment
Column: Amaze TH
Separation Modes: HILIC, cation-exchange, anion-exchange
Column Dimenstions: 4.6 x 100 mm 5 um, 100A
Mobile Phase: 80% ACN with 10 mM AmAc pH 4.8
Detection: ELSD
Sample: 0.3 mg/ml
Injection: 3 uL
Flow rate: 1 ml/min
Analytes
Class of compounds: Amino acid, Organic acid, Sugar, Supplement
Nature of compounds: Acidic, Hydrophilic, Neutral, Polar, Zwitterionic
Compounds: Succinic acid, Phenylalanine, Sucrose, Glycine, Aspartic acid, Raffinose
Ultra-Fast HPLC Separation of Underivatized Amino Acids in Reversed-Phase and Cation-Exchange Modes
Application description

Amino acids are essential components of numerous formulation. Health supplements can contain various amino acids and vitamins and require quantitation of each ingredients. Amino acids are very polar compounds with limited or no retention in reversed-phase chromatography. The most common approaches are reversed-phase chromatography with ion-pairing reagent and hydrophilic interaction chromatography (HILIC). Underivatized amino acids can be retained by combination of reversed-phase and cation exchange mechanism. These  two mechanism were combined in Coresep 100 core-shell mixed-mode column. Method can be used for fast analysis of underivatized amino acids without ion-pairing reagent. Depending on the pH of the mobile phase they can exist in acidic, basic or zwitter-ionic form. At lower pH, amino acids are basic in nature and have the highest hydrophobicity. Four amino acids (SER, GLY, ALA, VAL and DOPA) were retained and separated within 2 minutes on core-shell mixed-mode column. This high efficiency separation does not require special UPLC set up and can benefit from core-shell and mixed-mode approaches.

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: 3% ACN with 0.03% TFA
Detection: UV 210 nm, ELSD
Sample: 0.3 mg/ml
Injection: 10 uL
Flow rate: 1 ml/min
Analytes
Class of compounds: Amino acid, Catecholamine, Drug, Neurotransmitter, Supplement
Nature of compounds: Basic, Hydrophilic, Polar, Zwitterionic
Compounds: Glycine, Alanine, Serine, Valine, Dopamine