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 gradient utilities used to tweak your gradients in certain ways.

 

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Gradient utilities

The gradient helping tool can be split into three different tools. All of them are suitable for Type I projects only.

  1. Buffer gradient helper
  2. Gradient manipulator
  3. Transfer methods

1. Buffer gradient helping tool

Mobile phase buffer concentration is not usually considered as an important variable to optimize RPLC separations but is a critical variable for mixed mode stationary phases. If you are dealing with a separation problem requiring this type of columns, buffer concentration should be handled conveniently.

Mchrom Scout is able to simulate buffer concentration gradients concomitant with modifier gradients or/and temperature gradients in LC. This ability offers increased flexibility and added opportunities to find a satisfactory solution to your separation problem.

Buffer concentration gradients are commonly expressed in terms of the variation of buffer total concentration in the mobile phase so an instrumental system allowing the independent management of more than two solvent reservoirs is the choice for this type of runs.

The simplest method involves the use of three solvent reservoirs containing the modifier, pure water and a rather concentrated buffer solution having the desired pH.

In this way, the programming of simultaneous modifier-buffer concentration gradients can be easily performed. this is due to the total buffer concentration in mobile phase controlled via the proportion of buffer reservoir liquid included in the mobile phase whereas the modifier proportion is defined by the proportion of organic solvent reservoir and water proportion fulfills the condition A%+B%+C% = 100.

Translating the gradient program expressed in terms of buffer concentration and modifier percent into a sequence of lines defining the elution program in the solvent management systems is a quite simple task for any minimal experienced chromatographer and can be made even simpler using a worksheet. However, a critical step is defining the required buffer concentration in the buffer reservoir.

An incorrect calculation of this parameter would complicate or even negate the gradient programming. A new buffer bottle containing a different concentration should be prepared capable of executing some gradients. These operational failures can be avoided by using the Mchrom Scout buffer gradient helping tool.

 

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Initially this tool expects you define the total concentration of the dissolution in the buffer reservoir and also the assignment of solvent bottles to each mobile phase component (to avoid mistakes at the time of transposing the elution program to the chromatography software). Now, simply input the modifier program conditions desired (initial, final % and runtime) and the initial, final buffer concentrations required by the elution program and the corresponding runtime. The system will translate these values into buffer bottle percentages in the mobile phase. When finished, click on the Plot button and see one of the possible results:

Case 1. A feasible gradient has been defined.

Case 2. An non feasible gradient has been programmed.

In the first case, the elution program will be drawn in the graph and written to the gradient table. The graph permits the tracking of the gradient program and checks that the desired elution program has been effectively programmed by repositioning the mouse crosshair over the graph. The program table can be used directly to transpose the elution program to the instrument software in order to define the processing method or the sample set.

 

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If an non feasible gradient has been programmed, the program will alert you and the new plot will not be drawn.

 

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It is easy to detect when an non feasible gradient has been programmed because the proportions of water in the mobile phase become negative. Providing you have not made any errors in defining the gradient settings, the problem will be related to the total concentration in the buffer reservoir which appears too low to allow the gradient program to be executed. Thus, you simply increase the total buffer concentration value in the corresponding control to reach a feasible value of water % in the dialog form. In this way, you can preview your requirements before preparing the buffer solution avoiding erroneous mental calculations or assumptions.

 

2- The gradient manipulator

The gradient manipulator is a convenient tool allowing the simulation of any type of separation (isocratic or gradient of any shape) in response to your instructions. Thereby performing an efficient “what if?” process to allow testing your hypothesis and conclusions independently of the automatic default optimization processes performed by Mchrom Scout.

This tool is self-contained in a page control that includes:

  1. a) An editor page
  2. b) A chromatograph graphics page
  3. c) A data page

The editor page is the heart of the tool and includes several sub-pages to simplify the programming of the different types of gradients as well as the optional temperature and/or buffer gradient programs. also the modifier composition and pH if these variables are active in the project. Furthermore instrumental parameters can be changed here, so you can simulate chromatograms produced by instruments other than the one used to develop the retention model. This provides the maximum flexibility.

 

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A plot of the gradient program will be updated during your editing to verify the gradient you are programming is exactly the one you need. Here, the temperature program is defined by the blue plane in the gradient design space. Inside this plane, the modifier gradient is plotted (red line), as shown in the above image.

Once you are happy with your gradient program, simply change to the ‘Chromatogram’ tab (or to the Data tab) to see the result of your settings.

Here the gradient shapes (if temperature or buffer gradients have been defined additionally to the modifier gradient) are superimposed to the chromatogram to help in interpreting the influence of the programmed changed in the separation. The data page shows the retention values for peaks and the gradient program table.

 

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3- Transfer methods

Developed separations need usually need to be transferred to other instruments and/or column geometries. The need of transfer methods helping tools has been made clear after the irruption if U(H)PLC in the market and, currently a lot of commercial and free tools are available to this end. Mchrom Scout also includes a convenient tool for transfer methods. Mchrom Scout is superior to other existing applications, having developed a retention model. the transfer process does not just provide a “recipe” of the gradient corresponding to the new columns and/or instrument but also a view of the separation that can be expected in these new conditions. Thus providing an additional opportunity to adjust your separation to optimize your results.

In Mchrom Scout, the transfer methods utility starts from any simulated chromatogram active in the application (both chromatograms produced by the automatic optimization processes or from the gradient manipulator can be accessed for transfer), so the first step is to select any of these existing chromatograms.

 

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Following your selection, the system will recover all the information about that particular chromatogram as shown in the image bellow (left side), including the data corresponding to the instrument and column used to produce the model. Now you can alter these settings (e.g. in the way shown in the right side of the image). When you are happy with the changes, click on the (Scaled/Matched) transfer button to produce a simulation of the chromatogram in the transferred system.

 

 

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Depending on the state of the “Delayed injection available” checkbox (which, of course is dependent on the instrumental system applied) the application will perform a Scaled (the default option if possible) or a Matched transfer. The first option uses the usual rules accepted for geometric scaling transfer. However, not all instrumental settings allow this scaled process if delayed injection is not available. In those infrequent cases a process of re-optimization will be performed in order to ensure that the separation in the new instrumental system will be as much similar as possible to the current one.

The original and the transferred elution programs are provided in the tables tab to allow completion of the transferring process.

 

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Elution time can be adjusted by operating the flow rate settings manually as shown below.

 

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Thus, transfer methods tool in Mchrom Scout outperform existing applications with its additional options (runs in cases the standard geometric transfer cannot be applied and gives a view of the expected chromatograms giving options to adjust manually the separations before running true experiments with the new system).

 

 

 

 

 

 

 

 

 

 


Last modified: April 6, 2017 by Enrique Sánchez