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, 4.6×150 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

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Condition 2
Column Primesep C, 4.6×150 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 Column

Primesep D

The Primesep family of mixed-mode columns offers a wide variety of stationary phases, boasting unprecedented selectivity in the separation of a broad array of chemical compounds across multiple applications. Corresponding Primesep guard columns, available with all stationary phases, do not require holders. SIELC provides a method development service available to all customers. Inquire about our specially-tailored custom LC-phases for specific separations.

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Primesep C

The Primesep family of mixed-mode columns offers a wide variety of stationary phases, boasting unprecedented selectivity in the separation of a broad array of chemical compounds across multiple applications. Corresponding Primesep guard columns, available with all stationary phases, do not require holders. SIELC provides a method development service available to all customers. Inquire about our specially-tailored custom LC-phases for specific separations.

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Application Analytes:
Methylene Blue
Application Detection:
ELSD Detection
UV Detection
SIELC Technologies usually develops more than one method for each compound. Therefore, this particular method may not be the best available method from our portfolio for your specific application. Before you decide to implement this method in your research, please send us an email to research@sielc.com so we can ensure you get optimal results for your compound/s of interest.