Separation type: Bridge Ion Separation Technology, or BIST™ by SIELC Technologies

4′,6-diamidino-2-phenylindole, also known as DAPI, is a popular fluorescent dye used for DNA staining and fluorescence microscopy. Using SIELC’s newly introduced BIST™ method, DAPI can be retained on a positively-charged anion-exchange BIST™ B+ column. 

There are two keys to this retention method: 1) a multi-charged, negative buffer, such as Sulfuric acid (H2SO4), which acts as a bridge, linking the positively-charged analytes to the positively-charged column surface and 2) a mobile phase consisting of a majority of organic solvent (such as MeCN) to minimize the formation of a solvation layer around the charged analytes. Utilizing a step gradient to switch to a completely aqueous MP after 2 minutes allows for retention to occur while also preventing the method from being too long. Using this new and unique analysis method, DAPI can be separated, retained, and UV detected at 345 nm.

High Performance Liquid Chromatography (HPLC) Method for Analysis of DAPI

Condition

Column	BIST™ B+, 4.6×150 mm, 5µm, 100A
Mobile Phase	Gradient MeCN
Buffer	H2SO4 – 0.2%
Flow Rate	1.0 ml/min
Detection	UV 345 nm
Retention time	4.01 min

Description

Class of Compounds	Dye
Analyzing Compounds	DAPI (4′,6-diamidino-2-phenylindole)