The Advantages of a Targeted 2D LC Approach

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Courtesy of Courtesy of Waters Corporation

Webinar Highlights | Focus on the Relevant: The Advantages of a Targeted 2D Chromatography Approach

Hi folks, I’m Daniel Root, Senior Product Marketing Manager for Multidimensional LC Systems at Waters. I recently conducted a webinar that addressed multidimensional liquid chromatography – an exceptional tool for the improvement of characterization and separation of highly complex and challenging samples.

We discussed the fundamental aspects of multidimensional liquid chromatography, including the advantages and benefits of more targeted methodologies, such as heart-cutting, in relation to comprehensive LC x LC. In addition, we discussed a technique called at-column dilution. This is a Waters-patented technique that can be used to enhance the chromatographic performance, peak shape, and sensitivity of multidimensional analyses.

If you missed it or would like to revisit any of the subject matter, you can view the slides from the webinar or watch the webinar on demand.

We had a very engaged audience with many great questions. Here are some of the highlights from our Q&A session:

Dan, in your travels, how are people using 2D LC?

Generally, we see small molecule folks working to collect MS data from compendial HPLC methods and those working with large molecules applying 2D to automate analyses between different modes of separation such as SEC to IEX, or affinity to SEC, etc. Generating MS data is also common in the latter group.

In what industry areas are 2D LC techniques being used?

We are seeing 2D techniques being used in pharmaceutical life sciences, food and environmental science, and even the QC environment and non-regulated clinical laboratories. Users have definitely tuned in to the benefits of 2D approaches.

Is comprehensive and heart-cutting 2D LC the only 2D approaches?

While these two approaches are most common in the 2D world, and quite useful, there are other techniques associated with 2D systems. Multidimensional systems are generally made up of multiple pumps and multiple valves and can be used for trap and elution methodologies, online SPE, and increasing analytical throughput. We prefer to have our system fit the application need rather than offering a fixed inflexible configuration.

Can this approach be used for SFC?

The answer is a qualified yes. While there are researchers working diligently and succeeding in performing SFC-based 2D analyses (such as SFC/RP), there are no commercial products available at this time. Robustness and overall performance issues are still present in these type of experimental systems.

Is it more important to optimize your 2D separation or your 1D separation before the 2D?

That will depend upon whether you want to perform a comprehensive 2D analysis or a targeted heart-cut. In the former case, we usually start with the development and optimization of the second dimension separation. In comprehensive 2D LC this second dimension run time will determine how frequently you can sample the first dimension and will affect the parameters of the interface and the first dimension analysis. In heart-cutting, the first dimension separation will be optimized first. Often you are starting with your legacy HPLC method in these cases and optimization either isn’t allowed or isn’t necessary.

How do you deal with multiple heart-cuts to address more than only one target? Do you need to run a new analysis for every cut? You don’t want to mix those cuts together anymore before starting the 2nd dimension.

I strongly prefer to take one cut with one injection. If multiple cuts are required I recommend multiple injections. The primary reason is performance. It is really as simple as that. Variability in retention time and area for second dimension peaks in loop-based; multiple heart-cutting systems can be intolerably high, in my experience. Instead, a single heart-cut with at-column dilution optimizing the second dimension separation will deliver highly precise results. I would refer you to the webinar slides on performance.

How do you ensure that you really trap your whole peak? Unlike in online SPE you cannot just easily condition your trap column…?

Actually with a multiple pump, multiple valve-based 2D system that is precisely what you can do. The plumbing configuration can assure that all columns and traps are conditioned and equilibrated properly for optimal performance. As for the first part of your question, I’d have to say that chromatography is chromatography whether you are using one column, or two, or two with a trap.

How do you have confidence that your analyte(s) of interest are retained reliably on a single dimension analysis?

Just like with 1D separations, you will characterize the optimized conditions for retention prior to running your 2D analysis.

What approach do you do with complex samples?

To decide this, you will need to be clear on what kind of information you wish to glean from your sample. If you have known specific analytes in mind, then a targeted method will be best. If you have only a basic idea of what might be in your sample and simply need to characterize everything, then a comprehensive approach will likely give you what you need.

Are there any limitations to at-column dilution?

Occasionally, you may come across a circumstance where the first dimension flow is so high that in order to dilute to the desired level you may need a dilution flow rate that is higher than the flow capability of the dilution pump. In these cases, the flow rate of the first dimension may require adjustment.


Thank you for your time and excellent questions!

You can also learn more about the Waters 2D LC technology for UPLC/UHPLC separations on the Waters’ website.

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