For several reasons the storage of 100% pure solvents in the reservoir bottles of an HPLC system can be a problem. Pure acetonitrile, in particular, affects the system by adsorption of polymers from transmission lines resulting in sticky surfaces that inhibit the opening and closing of the valves. Pure water or buffers are susceptible to the formation of algae or fungi. For these reasons, it is recommendded to use premixed solvents with water in acetonitrile to avoid polymer adsorption problems and organic solvents, such as acetonitrile or methanol, in buffer to prevent micro organism’s formation. Pre-filtering this mixture will also have the effect of lowering the possibility of buffers precipitating when mixed in-line with organic solvents.

Another advantage of premixing solvents is related to the gradient slope. When a smooth slope requires the mixing system to create small changes, for example 0.33% of solvent A per minute, this may not be possible with older, low pressure gradient mixing units with a typical precision of ± 0.3 – 0.5%. The example in the box illustrates that the use of premixed solvents results in a steeper slope for the programming of the system, which will provide a higher precision, and robustness of the analysis.

Whilst it is more convenient to perform method development with pure solvents, but once the method is ready for routine use, the user has to do some annoying calculation work, to avoid the problems mentioned above for pure solvents. To make this work easier, we have included an Excel tool in this article, which allows quick and convenient translation of gradients.

In the top box single concentrations can be calculated. White cells are for input of values or names, blue cells contain calculated values. The values for the required concentration of A in the eluent, the name of the solvent A and the concentration of the solvent A in the two reservoir bottles can be entered. The result in the red cells gives the proportion of the two pumps that are necessary to create the wanted concentration of solvent A in the mobile phase.

To make gradient calculations more convenient in the box at the bottom you can find a table where the required values for solvent A (solvent B is calculated automatically) and the concentrations of solvent A in the reservoirs can be entered. The tool will calculate the required proportions of the pumps with the given concentrations of solvent A in the reservoirs and display them on the same line in the red cells.

Click here to download the Excel tool.

For more information please contact Dr. Wolfgang Schräder. YMC Europe GmbH, Phone: +49 (0)2064 427-290, Fax: +49 (0)2064 427-222 or visit www.ymc.de