Trends in 2-D Liquid Chromatography

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Dwight Stoll is an associate professor in the Chemistry Department at Gustavus Adolphus College in St. Peter, Minnesota, where he teaches quantitative and instrumental analysis courses in addition to directing a vibrant research program involving mainly undergraduate students. He holds bachelor of science degrees in plant biology and biochemistry from Minnesota State University, Mankato. Professor Stoll is the author or co-author of 49 peer-reviewed publications and two book chapters in the area of separation science, is a named co-inventor on four patents, and has instructed numerous short courses in two-dimensional liquid chromatography (2D-LC).


Q: What do you use 2D-LC for in your work?

A: In my laboratory, we use 2D-LC for separating both complex materials and [for] mixtures that are not complex per se but contain pairs or groups of molecules that are inherently difficult to resolve. Examples of complex mixtures include tryptic digests of proteins, plant extracts, and natural water samples. Samples in the difficult-to-resolve category often contain isomers—regioisomers and stereoisomers. I believe that conventional liquid chromatography, 1D-LC, will continue to be a dominant technique for a long time. But it has limitations, and in most cases 2D-LC can be an effective and efficient way to solve problems that cannot be easily addressed by 1D-LC.

Q: How many people work in your lab?

A: We don’t have a graduate program at my institution so I work exclusively with undergraduates and postdocs. Typically, on average, I have about six undergraduates working with me and then one postdoc or technician.

Q: What are some of the recent trends in 2D-LC?

A: Right now there is a lot of growth in the adoption of 2D-LC in a variety of industries, but probably the most notable one is in the pharmaceutical industry. Researchers in pharma and biopharma today can buy commercially available instruments for 2D-LC that simply weren’t available five years ago. These instruments are both sophisticated and relatively easy to use compared with where the technology was at five and certainly ten years ago. Even more exciting at the moment is the fact that some groups in pharma are pushing methods involving 2D-LC separations into QC labs where the methods are highly regulated and must be very reliable. This means that the community is becoming increasingly confident in the technology and is committed to it for the long run.

Q: What have the changes in 2D-LC meant for your work?

A: Ten years ago most of my work on 2D-LC was perceived as being pretty “far out,” meaning that people could not imagine our results having an immediate impact on their work. Today, with so many more users of 2D-LC, the community is much more interested in the research going on in academic laboratories. My group is very collaborative, and we have been establishing productive collaborations with different groups in pharma to focus our work on problems that will have an immediate impact in the use of 2D-LC for pharma applications.

Q: What are some of the applications you’re working on with those groups?

A: The place where we’re getting the most traction with those interactions is in the biopharma space, so antibodies, therapeutics, proteins—things like that. Historically, I’ve had a really hard time establishing those kinds of relationships, but not because there isn’t interest. It mostly comes down to legal issues and the extent to which [biopharma companies] tend to be very secretive about their molecules, which is understandable. I think because in the biopharmaceutical spaces there’s so much energy, so much interest in moving ahead quickly, there’s been more willingness to figure out how to make [collaborations with academia] work.

Q: What are some of the most common challenges you encounter when working with 2D-LC?

A: Lately in my research talks and conversations with people, I have been referring to the second-dimension part of 2D-LC systems as a “strange place.” It is strange in the sense that the conditions we use there and the kind of results we see are often very different from what we encounter in conventional liquid chromatography. For example, in some situations we are interested in doing extremely fast gradient elution separations in the second dimension on the order of 15 seconds. Doing this effectively demands hardware that is sometimes difficult to find. For example, many HPLC columns are available only in lengths longer than 3 cm. If we want a 2-cm-long column in a particular chemistry, we are stuck.

Q: How do you handle those challenges?

A: We work collaboratively with instrument and consumable vendors to encourage them to expand the scope of their offerings to include items and product characteristics that might not be so valuable for conventional LC but are very valuable for 2D-LC. In this we have a classic chicken–egg problem—if users of 2D-LC cannot buy the equipment they need, they will not be able to develop the applications they need, and if they don’t develop those applications, then there is no market for the vendors to sell to. So we see that it is a slow process, but at least it is moving now.

Q: What do you enjoy most about working with 2D-LC?

A: Good question. My educational path has involved elements of engineering, biology, and chemistry. I find that working on 2D-LC allows me to combine all of those interests in a way that helps us push the technology and the state of the art forward. And it is very rewarding to see the adoption of 2D-LC across different industries picking up speed now.

Q: What applications of 2D-LC do you find most interesting/ promising?

A: In my laboratory we have made a pretty big shift over the past couple of years to focus a lot of our attention on applications in biopharma (e.g., antibodies, peptides). This is a research space where high-resolution separation methods are badly needed, and the molecules often lend themselves to very effective 2D-LC separations. I believe great strides will be made in this area over the next few years.

Q: How do you expect 2D-LC to change in the future?

A: I think the trajectory will be similar to what we’ve observed with LC-MS. In the early days of LC-MS, the instruments were not very robust, users were experts, and the software was not well developed. Now, many LC-MS instruments are used by people we would not call expert mass spectrometrists, because the hardware has become more reliable and easier to use and the software has become smarter and taken a lot of decision-making burden off the analyst. I think we will see similar things with 2D-LC; the hardware will become more effective, more reliable, and easier to use. And the software will do more to help the user develop and use methods, rather than getting in the way of good ideas. I believe the future is bright.

Q: In your experience teaching courses in 2D-LC, what are some of the most common problems people encounter when they’re first learning the technology?

A: I think the single biggest thing is that people new to 2D-LC can feel overwhelmed by the complexity of the technique. There are many more variables to consider in method development and I think people have a difficult time knowing where to start.

Q: How can they deal with those issues?

A: We have known for some time that this is a problem, and we are addressing it in two ways. First, we are constantly thinking about how to improve the hardware and software for 2D-LC to make it easier to use, and thinking about how to develop method development strategies that people can apply broadly. Second, we know that education is very important, which is why we have developed the short courses and teach them every chance we get.

Q: What advice do you have for labs that are looking at adding 2D-LC?

A: Try to avoid reinventing the wheel. There are really great examples in the literature [from] the past five years or so of very high-performing 2D-LC methods in a variety of application areas. Try to learn from those methods and adapt them to your needs. Also reach out to experts in the community. The people I know who have a lot of experience with 2D-LC are generally very interested in talking with people about their applications—I know I would certainly be happy to talk with anyone interested in getting involved in 2D-LC.

Q: You recently won a Thought Leader Award from Agilent for your work with 2D-LC in biopharmaceutical analysis. How do you feel about that?

A: It’s great. I think it really speaks to their interest as a company in terms of supporting research on 2D-LC generally. Obviously, the project that we’ve designed around that [award] is applications of 2D-LC in biopharma, but they’re putting a lot of financial support behind [2D-LC research in general] and I think it speaks to their confidence in the future of the technology. For me personally, it’s a huge win for my research program and will provide really great support, but, like I said earlier, it’s rewarding to see the technique gather more and more interest, in a broad sense, across the industry.

Categories: Ask the Expert

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Published: July 13, 2017

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