Which standard operating procedure in a preparative chromatography lab is more efficient: routinely running generic full-range gradients to purify mixtures, or taking the extra time and trouble to develop focused gradient methods? The answer may depend, in general or on a case-by-case basis, on how long it takes to develop the method, the purification run time, the quantity of sample and solvent consumed, and your output quality requirements (level of purity, yield).
Simple full-range (0 to 100%) gradients require minimal time to develop and are useful for mixtures with a wide range of structures. To improve resolution the analyst may extend the run time to 'flatten' the gradient. The benefit of not having to develop a more detailed method, however, is substantially offset by the longer run time and added solvent required to achieve resolution between compounds.
Consider as well that in many prep LC applications the goal is not to resolve multiple compounds, but instead to separate one target alone, as quickly as possible, and with the highest practical purity and yield. Optimizing a prep LC method for single-target purification takes additional effort, which is often justified by faster purification, reduced solvent consumption, and higher productivity based on the ability to load more compound per run.
When comparing the pros and cons of generic vs. focused gradients, it helps to envision the changing behavior of a compound during a 0% to 100% gradient. Once the injected sample has adsorbed onto the stationary phase, and before the target compound has fully eluted, it will be in one of three states (below) in which k* represents the retention factor under specific gradient conditions.
- Strong retention on the stationary phase; compound not moving (k* >> 10)
- Weak retention on the stationary phase; beginning to travel down the column (1 < k* < 10)
- Zero retention on the stationary phase; compound traveling at solvent front (k* < 1)
These three pre-elution states each correspond to a specific %B zone within the gradient. There is a fourth zone post-elution where the process continues to run while the compound is already off the column.
From a purification standpoint, the simplest case would one in which all non-target compounds are completely retained while the target is fully eluted. The reverse occurs when the target compound is retained on the column, while the impurities fully elute. The target is then eluted using a second strong solvent, that is, a 'catch and release' purification.
The most difficult, by contrast, would involve two or more compounds having weak retention in the same %B range, in which case they will easily co-elute unless an optimized gradient is employed.
Generic gradients are relatively inefficient as compared to focused gradients. In a generic method, the most effective segment of the 0-100% gradient range is the 5-15 %B portion in which the k* value for the target compound is between 1 and 10, with the compound actively migrating down the column. In a generic gradient, therefore, 85% to 95% of the run is wasted because the compound is a) still sitting at the head of the column and not moving at all; or b) moving with the solvent front and having no further interaction with the stationary phase; or c) already eluted and collected into your test tube.
Focused gradients on the other hand are faster and less wasteful. The bright idea behind the focused gradient approach is to simply increase the time the target spends in conditions where k* is between 1 and 10 (the most productive separation zone), while decreasing the time spent in all other %B zones where it is either being fully retained or traveling at the solvent front. Once the optimum %B range for a target compound is identified, such as by a scouting run or other means, refining the prep LC method to focus primarily on that zone produces significantly faster separations with better resolution between closely eluting compounds.
Assuming focused gradients are better in every important way—speed, accuracy, economy, and even environmentally greener through reduced solvent consumption, what should be done to promote wider adoption of the technology? At Teledyne ISCO we believe this can be achieved by removing the most significant hurdle to adoption, the time and expertise needed to develop focused gradient methods. This has long been the focus of our technical support and product development efforts. For example, there are several calculation methods users can employ to arrive at an optimized prep LC method based on minimal data about the target compound's properties or behavior. Algorithms such as Accelerated Retention Window (ARW), Time on Target (ToT), and First Time Right Time are very well summarized in my colleague Jack Silver's article [1] published in ACS Combinatorial Science.
Then in 2019, to make the process even faster and easier by avoiding all manual calculations, and building further upon the work of Jack Silver, we released our automated Focused Gradient Generator tool [2] within the PeakTrak software on the ACCQPrep HP150 system. This tool allows a user to perform a scouting run on the HP150, select the peak of interest, and have PeakTrak respond instantly with an optimized focused gradient. And it automatically scales methods to larger prep columns of the same chemistry. With the improved resolution of the optimized method, higher loads can then be injected for further runs on the same prep column.
So what could be easier? Well, what if the scouting run did not have to involve a prep column, and did not have to be run on the HP150? Can we take the retention time from a scouting run performed on an analytical U/HPLC system, enter it into the ACCQPrep, and have PeakTrak automatically generate an optimized focused gradient? Yes, with a little extra software development, and our updated PeakTrak software version 4.2.68 or later [3] now provides this capability.
In order to use this feature, the analytical system must be calibrated (correlated) to the ACCQPrep using a standard test mix and the instructions in Technical Note 52 [4]. Note that calibration parameters are different for HPLC vs. UHPLC systems, and that analytical and prep columns require matching stationary phases, and the runs should use the same mobile phase and modifiers.
Once the calibration is complete, PeakTrak will automatically scale the prep method for any column size you select from the screen. This works even if you are using non-RediSep® columns, in which case an additional simple step (included in TN52) is required to calibrate the prep column.
Once an analytical system is calibrated to the ACCQPrep, focused gradients for prep methods are generated just by entering the retention time from the analytical run into the ACCQPrep.
Optimized purifications are more accessible than ever, now that there is such an easy way to derive focused gradients from your analytical system or the ACCQPrep. Should any other reason remain for not using optimized methods more often in your prep lab, just send me an email and we will solve it. And please use the Submit Feedback button as well to let us know your thoughts, questions, or comments, which we consider vital to our ongoing product development efforts.
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