Raffinose

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