Better, Faster, when closely eluting peaks present a purification inefficiency.
Gradient elution employs a programmed change over time in mobile-phase composition (weak to strong) during a separation. A Linear gradients (most common) is a constant change in mobile phase composition over time. A step gradient is a mobile phase composition that targets the retention of the specific compounds in your sample.
In many cases, a simple linear gradient is enough to separate your compounds well.
BUT WHAT IF THAT DOES NOT WORK?
Time to Use a Step or Focused Gradient!
A step gradient is comprised of a series of isocratic holds with different mobile phase combinations. These “steps” maintain the solvent composition for portions of the separation that need increased resolution, without increasing overall separation time, and also potentially reducing solvent use.
Examples of Gradients
In difficult separations, a step gradient can shorten the run time and reduce the volume between eluting species all while improving the resolution of closely eluting peaks. It can also be useful in isolating the peak of interest, from other components within the mixture, overall shortening the run. These methods are greatly important on the industrial scale as well.
Isolation of a Peak of Interest
It would seem intuitive to place an isocratic hold into your run based on the elution time of the closely eluting or peak of interest from the resultant chromatogram of your scouting linear gradient run, but this is not the case. By the time these peaks are detected they have already exited the column and are in the detector. Any change to the gradient at this point will be AFTER the actual separation of the closely eluting species has already occurred.
To properly set an effective Step gradient you need to know the system volume as well as the column volume. This will provide the offset volume between the detector and the start of the gradient. Using this information and your flow rate, you can calculate the actual amount of strong solvent that eluted the peak at the actual time the peaks were resolved, not detected.
A good starting point is to use a 5% below the expected percentage of your strong solvent that will elute the peak of interest, and finish the shallow, focused portion of the gradient with a percentage of B that is 5% above the percent of B that eluted your peak in the scouting run.
Remember the hold time for the Step/Focus must be calculated taking into consideration the number of column volumes at your flow rate used to elute the peaks of interest from your scouting run. We would recommend that column volume need to be established using the apex of your peak of interest, but adding additional column volumes to ensure that the Step begins prior to the peak and the end of the Step is after the termination of the peak of interest.
Many of today’s Automated Flash Systems have software with developed algorithms that can automatically calculate the needed changes, including times to establish a Step/Focus gradient from a previous run or TLC inputted data.
Our SepaBean Automated Flash System is an example of one of those intelligently capable instruments that can automatically suggest a column as well as a gradient based on inputted TLC or HPLC data from the user. It also can program in the Step gradients as discussed above from user input.
This intelligent system goes far beyond these important characteristics, here are some other features of the SepaBean Automated Flash System.
