Analysis of Methylene Blue Purity in Reversed-Phase Cation-Exchange and Reversed-Phase Cation-Exclusion Modes

Methylene blue is a heterocyclic dye with a wide range of use in biology and chemistry. Purity of dyes rarely exceed 70% and separation of methylene blue and related impurities is required. Two alternative methods for purity analysis were developed on Primesep C and Primesep D mixed-mode HPLC columns. Primesep C is a reversed-phase cation-exchange (anion-exclusion column) column and Primesep D is reversed-phase anion-exchange (cation-exclusion) column. Various buffers can be used for the method. Depending on the buffer, method can be compatible with UV, LC/MS and prep chromatography. Method can be used for prep separation of methylene blue and related impurities.

Condition 1

Column Primesep D, 4.6x150 mm, 5 µm, 100A
Mobile Phase Gradient MeCN - 5-20%
Buffer Gradient NaH2PO4 pH 2.5 70-20mM 15 min
Flow Rate 1.0 ml/min
Detection UV, 300 nm
> The antibiotic norfloxacin is a first generation fluoroquinolone. It is effective against both gram positive and gram negative bacteria. Analysis was done on Obelisc R and Primesep D.  

Condition 2

Column Primesep C, 4.6x150 mm, 5 µm, 100A
Mobile Phase MeCN - 35%
Buffer AmFm pH 3.5 - 35 mM
Flow Rate 1.0 ml/min
Detection UV, 300 nm
 

Description

Class of Compounds  Duy, Hydrophobic, Ionizable
Analyzing Compounds  Methylene Blue
 

Application Analytes:

Methylene Blue