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.
| 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.
| 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 |
| Class of Compounds | Duy, Hydrophobic, Ionizable |
| Analyzing Compounds | Methylene Blue |
Application Analytes:
Methylene Blue