CombiFlash Gradient Methods

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Courtesy of Teledyne ISCO

Abstract

Purifying compounds require two processes: the actual purification and drying the sample afterwards. Several gradient methods were evaluated on a reaction mixture to evaluate run time and number of fractions to collect the desired compound. Reducing the volume of fractions is important to reduce the time required to dry the purified material. On large runs, drying time may be equal to, or greater than, the purification time. An optimized step gradient was found to be most efficient for repeated purifications, but required several runs to optimize. The most efficient gradient for general use was programmed into PeakTrak®. A comparison was also run between various automated gradients and a manual method: Dry Column Vacuum Chromatography. The PeakTrak Gradient Optimizer was found to be efficient when purifying compounds that eluted close to each other on TLC plates.

Introduction
There are several different gradients that can be run on a CombiHash system:

  • Isocratic: The solvent composition remains the same throughout the run.
  • Step Gradient: The strong solvent changes abruptly to a higher concentration.
  • Linear Gradient: The concentration of the strong solvent gradually increases over time. The slope of the linear gradient can be changed to vary the resolution between eluting peaks.
  • Mixed Gradients: These are a combination of linear gradients with isocratic holds. They can be generated with the Gradient Optimizer on the CombiFIash system.

Each gradient type has advantages and disadvantages which will be discussed in the Experimental and Results section.

Experimental and Results
Synthesis of 3-(2-nitrophenyl amino) propionitrile

2-Nitroaniline (304 g) was dissolved in 1500 mL reagent alcohol. Triton B (45 mL) was added and the mixture was heated to reflux. Acrylonitrile (420 mL) was added with stirring. The mixture was stirred overnight at reflux. The alcohol was evaporated to yield -550 g  of a tarry mixture. A portion of this mixture was loaded onto silica by dissolving it in dlchloromethane/meth-anol/water and adsorbing onto silica (I part reaction mixture to 4 parts silica. 20% load on bulk RediSep*silica (PN 60-3874-091)) and the solvents evaporated. This sample was used for all experiments (except where otherwise noted) in this application note; 4.0g was used for each experiment (0.80g reaction mixture, 1.0% load on an 80g RediSep® silica column PN 69-2203-380).

All purifications were run on an Combi/7os/f® Rf-200 system (PN 68-5230-008) using solvent A= hexanes; solvent B= ethyl acetate. Peak collection used 254 nm.

Isocratic Isocratic is the easiest method to develop and is also the easiest to run because no special equipment is required if the solvent is premixed. Isocratic methods are best run on simple mixtures that are well resolved.

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