Mchrom Scout provides several tools designed to help the chromatographer to obtain a more efficient method development for RPLC separations. These tools are under the ‘Utilities’ tab and are divided into three main groups of tools:

  • Gradient utilities
  • Pareto Optimality
  • Resolution maps

 

These tools are available via the Utilities menu when you require them. They are organized for easy use as a function of the project type selected as well as during the current stage of specific optimization process.

This tutorial we will show you one of these three utilities tools, the resolution maps, used to tweak your gradients in certain ways.

 

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Resolution maps helping tool

Resolution maps have been the most commonly used criteria and decision making tool along the years for computer assisted method development in chromatography. A resolution map is a graphical representation of the critical resolution (the resolution of the worst resolved pair of peaks in the chromatogram) vs. the control variables used to develop the optimum separation. Usually, 1-3 control variables were considered, being the gradient time, temperature or pH and the solvent mixture composition in the mobile phase, the common choices.

Although in Mchrom Scout this is not the default strategy, a powerful resolution maps helping tool has been implemented.  Resolution maps in two or three dimensions can be configured easily not only based on the main optimized variables. All variables affecting the separation (e.g. the curved shape of the gradient, or the possibility of performing simultaneously temperature and modifier gradients), that can be calculated for a given project type, are available to configure the resolution map.

Assume you are developing the separation of a mixture of 15 components using the gradient time as the only active variable. Using resolution maps what you can expect as the result of your process is a graph similar to the one shown below, representing the critical resolution as a function of the only variable (tg) and the system will select as optimum the separation corresponding to tg = 26 min for a gradient developed between 5-95% of acetonitrile. All peaks in the mixture are visible although some of them lack sufficient resolution for quantitative purposes.

 

resolution maps helping tool

 

Mchrom Scout enables you to explore other gradient settings (e.g. different modifier percentage at the starting and ending points) in the search of a better resolution map or include in the study additional variables such as temperature, pH, etc.

Using Mchrom Scout, you can explore additional options by enlarging the dimensions of the resolution map. For example, you can see that using curve gradients no improvement in resolution can be obtained, although very similar results can be produced using curves 4 and 5 (WatersTM gradient programmers).

 

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Or you can confirm that the best results are produced by starting the gradients in 5% ACN by producing a 2D resolution map including the gradient time and the % ACN at the start as shown in the next graph.

 

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Now, you can select the best gradient slope by drawing the 2D resolution map including the tg and the final percentage of ACN as active variables.

 

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However, you can define directly the optimal slope and range for gradients by fixing time at the optimum value found initially (26 min) and using the initial and final percentages of ACN as active variables, as shown below:

 

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And, of course, you can explore all these three variables simultaneously by producing a 3D resolution map assigning a minimal value to the critical resolution.

 

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The optimum in that example corresponds to a linear gradient from 9 to 40% of ACN developed in 49 minutes. Thus, you can see that the Resolution Maps helping tool exceeds your expectations.

The greater the number of active variables included, the greater number of combinations producing resolution maps to help you find your optimal solution. However, the Rs map helping tool uses a grid search to produce the plots and find the optimum in each case, so the calculation time needed is somewhat higher depending on the number of peaks in the chromatograms.

Mchrom Scout uses alternative automatic optimization modes based on evolutionary algorithms that provide direct access to the optimum without the need of a manual developing process. The Resolution map helping tool was included and enhanced for those chromatographers used to this optimization strategy that prefer a manual process for searching this optimum.

 

 

 

 

 

 


Last modified: April 6, 2017 by Enrique Sánchez