Achieving Efficient Purification in the Presence of Closely Eluting Peaks
Gradient elution involves a programmed change in mobile-phase composition over time, transitioning from weak to strong during a separation. Linear gradients, characterized by a constant change in mobile phase composition over time, are commonly employed in chromatography.
A simple linear gradient is often enough to separate your compounds well. However, in situations where a linear gradient falls short, step gradients or focused gradients come into play. A step gradient is a mobile phase composition that targets the retention of the specific compounds in your sample.
Step gradients comprise a series of isocratic holds with varying mobile phase combinations. These “steps” maintain specific solvent compositions during critical phases of the separation that need increased resolution. This is achieved without increasing overall separation time, and step gradients may also potentially reduce solvent use.
Examples of Gradients
Image Credit: Sorbent Technologies, Inc.
Step gradients prove invaluable under challenging separations. These gradients reduce run times and minimize the volume between eluting species, all while improving the resolution of closely eluting peaks. They are also highly effective for isolating the peak of interest from other components within the mixture, ultimately shortening the process. These methods are essential on the industrial scale.
Isolation of a Peak of Interest
Image Credit: Sorbent Technologies, Inc.
When isolating a peak of interest, intuition might suggest introducing 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. However, this is not the case.
When these peaks are detected, they exit the column and reach the detector. Any adjustments to the gradient at this point will occur after the separation of closely eluting species has occurred.
The system and column volumes must be known to set up an effective step gradient. This information helps determine the offset volume between the detector and the gradient's start. Using this information and the flow rate, the amount of strong solvent that eluted the peak of interest can be calculated when the peaks were resolved, not detected.
A recommended starting point is to use a strong solvent percentage 5% lower than expected to elute the peak of interest. Then, it is recommended to finish the shallow, focused part of the gradient with a percentage of B that is 5% higher than what was used to elute the peak in the scouting run.
It is important to remember that the hold time for the step/focus must be calculated, considering the number of column volumes at the flow rate required to elute the peaks of interest from the scouting run. It is advisable to establish the column volume using the apex of the peak of interest while including additional column volumes to ensure that the step initiates before the peak and concludes after the peak’s termination.
Modern Automated Flash Systems often feature software with developed algorithms capable of automatically calculating the necessary adjustments, including times to establish a step/focus gradient from a previous run or TLC inputted data.
Sorbtech’s Sepabean Automated Flash System is an example of such an intelligent instrument. As discussed above, the system can automatically suggest appropriate columns and gradients based on user-provided TLC or HPLC data and program the step gradients.
Beyond these crucial functionalities, the Sepabean Automated Flash System boasts a range of additional features:
Sepabean, Automated Flash Chromatography Purification Systems
Image Credit: Sorbent Technologies, Inc.
- Mobile device wireless operation. The flexible wireless control method is especially valuable for separation experiments requiring protection from light or placement in an isolator.
- Power failure recovery. The software includes a built-in power-off recovery function, minimizing losses caused by unexpected power failures.
- Separation method recommendation. The software boasts an integrated separation method database that automatically suggests the most suitable separation method based on the key information inputted by the user. This capability thereby enhances work efficiency.
- Fraction collector. Tube racks with LCDs enable users to track the tubes containing collected fractions easily.
- Local network data sharing. Create a local area network with multiple instruments to facilitate internal data sharing and resource optimization in the laboratory.
- 21-CFR part 11 compliance. The control software complies with FDA requirements for system safety (21-CFR Part 11), making it an ideal choice for pharmaceutical R&D companies and laboratories.
- Smart purification system. This smart flash chromatography system includes an innovative, built-in separation method recommendation feature, enabling even novice users to harness the instrument’s capabilities and achieve results akin to those of experienced chromatographers.
- Smart purification with “Touch & Go” simplicity. A simplified UI (user interface) means a straightforward, direct, and easily understandable system that requires minimal training. This ensures all users, regardless of experience, can complete routine to complex separations easily and immediately.
- Built-in method database — knowledge retained.
- Unlike traditional flash instruments, the Sepabean™ T employs database and distributed computing technology to retain and share stored methods across a secured organizational network.
- Patented Sepabean™ T incorporates a built-in relational database for storing separation methods. Researchers can query existing methods or update new ones simply using the compound name, structure, or project code.
- Sepabean™ T is network-ready, allowing multiple instruments within an organization to establish a private channel. This enables the sharing of separation methods across the entire organization, granting authorized researchers access to and the ability to run these methods directly without the need for method re-development.
- Sepabean™ T can automatically discover and connect to peer instruments. Data is synchronized automatically once multiple instruments are connected, allowing researchers to access their methods from any connected instrument, regardless of location.
This information has been sourced, reviewed and adapted from materials provided by Sorbent Technologies, Inc.
For more information on this source, please visit Sorbent Technologies, Inc.