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David Hage, PhD, Discusses Trends in HPLC Columns

David S. Hage, PhD, is the James Hewett University Professor of Chemistry at the University of Nebraska-Lincoln.

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David S. Hage, PhD, is the James Hewett University Professor of Chemistry at the University of Nebraska-Lincoln. Dr. Hage’s general research interests involve the design and use of affinity-based separations in high-performance liquid chromatography (HPLC), capillary electrophoresis, and other systems for clinical, pharmaceutical, and environmental analysis. He is also interested in the use of affinity-based separations, both alone and in combination with mass spectrometry, as tools for personalized medicine, functional proteomics, and metabolomics. Dr. Hage received his PhD in analytical chemistry from Iowa State University and his BS degrees in chemistry and biology from the University of Wisconsin-La Crosse.

Q: What does your lab do?

A: My laboratory does research in the area of developing new types of chromatographic supports and methods, specifically for bioanalytical purposes. We specialize in high-performance affinity separations where we use biologically related molecules to capture and bind a particular compound or to study interactions in biological systems by seeing how an immobilized agent binds to another agent in a solution, such as in examining a drug-protein interaction or a protein-protein interaction. We do a lot of work, not only in developing those types of techniques but also in exploring new applications, new detection modes, and looking at the theory behind some of those methods.

Q: How many students/staff do you have in your lab?

A: My laboratory typically has eight to 10 graduate students in chemistry and perhaps one or two postdocs, and then usually two or three undergraduates in chemistry.

Q: What do you use HPLC for in your lab?

A: Most of our work involves using HPLC for either analytical work or developing new ways of looking at things like drugs, hormones, proteins, and biomarkers in biological fluids or environmental samples. We also use HPLC in a bit of a unique fashion to study interactions in biological systems by basically making a chromatographic system that’s a model for a biological system. For example, you can make a model of a blood-type system by immobilizing a protein from blood into an HPLC column and looking at how this protein interacts with things that might be transported by the protein in blood.

Q: What are the most common types of HPLC columns that you use for your research?

A: We make a lot of our own columns because in the area of affinity chromatography there are some available commercially, but a lot of the ones that we require are based on specialized proteins or proteins that are isolated from specific samples. So another thing we do a lot of is we take immobilized agents and we prepare them for use with various types of chromatographic columns and supports we want to study. We can then use these components to look at an interaction or to develop a new analysis method. We do everything from developing new immobilization techniques and characterizing immobilized biological agents to learning how to prepare various types of columns in unique formats—for example, microcolumns. Along with this, we also use traditional HPLC columns, based on ion exchange, size exclusion, or reversed phase chromatography. These latter columns are usually obtained commercially.

Q: What major changes has your lab recently experienced in terms of HPLC columns?

A: We’ve been working quite hard for many years to develop columns that contain immobilized biological agents that have high activity and are good mimics of the same agents in biological systems—for example, serum transport proteins. We have learned how to prepare these immobilized agents and place them into columns either in a chemically immobilized form or in an entrapped form where they’re still placed within the column but they can’t move out of the column. We then use these columns as a way to study biological systems. Some other work we’ve been doing in the past 10 or 15 years has been to learn how to make miniaturized affinity columns. Affinity columns by themselves are usually much smaller than what you’d see with other types of HPLC columns. Often a big column in our work might be only two and a half centimeters long, though right now we’ve been doing a lot of work with columns that are one centimeter or less in length and often only one or two millimeters thick. These columns are used to allow us to get into some unique regions of chromatographic behavior that you can’t get with traditional HPLC columns. So learning how to make those microcolumns and how to use their unique behavior are some challenges that we’re always facing.

Q: What are some of the other major challenges you face with HPLC columns?

A: We’re always looking for more efficient supports, especially those that could be used at high flow rates, so another area that we’ve been going into more over the past decade is working with monolithic columns, both organic-based and silica-based. For us, useful features of these monoliths are that we can make them with various sizes and shapes. These materials also have very low back pressures compared with particulate supports, so we can do very fast separations with them. That’s useful for some of our applications where we’re interested in doing work where the sample is in a column only for a fraction of a second. We do work with these and other materials in developing techniques where we can extract out a molecule, like a drug or a hormone, and do it on the time scale of maybe a hundred milliseconds.

Q: When creating HPLC columns for your lab, what criteria do you look for?

A: Besides considering things like the efficiency and the back pressure, one unique thing we have to think about when using immobilized biological agents is the pore size. In most traditional HPLC columns, like those used in reversed phase chromatography, the pore size may be only 50 to 60 Å or 100 Å. For us, we’re often dealing with a biological agent that is maybe 50 to 100 Å in size or larger, so the pore sizes on these types of columns would be much too small to allow us to get a protein onto the support and use a reasonable amount of the surface area. For us, having pore sizes that are more on the order of 300 to 500 Å can be quite important, and sometimes we even use pores of 4,000 Å, depending on how big of an agent it is that we’re trying to immobilize. For example, we’ve done work with lipoproteins, which can be quite big and require pore sizes of 1,000 to 4,000 Å. As you increase the pore size, you decrease the surface area, so there’s always a compromise between those two parameters in terms of how much agent you can place into the column.

Q: What resources do you find most helpful when it comes to creating your HPLC columns or when dealing with a problem with your columns?

A: We do a lot of characterization of our columns and supports—so back pressure and efficiency studies are important for that type of work. Those studies are pretty important for us to figure out how the columns are performing or might perform. And we also have various mathematical tools that we use to help characterize the columns. There are equations we can fit to the data to describe what’s happening and then predict how the columns might behave under other conditions—such as other sample concentrations or other flow rates. We do a lot of that work initially with test columns and then, once we have that behavior better established, we can make more of the same type of column or we can improve the design to go to the next phase of work. Of course, once we get a method validated by comparing our technique with other methods, we can go on and explore some unique applications for it.

Q: Do you have anything more to add?

A: Besides doing research, my lab is a training ground for students who will be going on and doing research in an industrial setting, a government facility, or a research lab. In many ways, a lot of what the students are learning here is basic HPLC—learning how to take care of systems, how to evaluate columns, and how to evaluate methods. We use those basic tools in a different setting or in different applications than most people do, but it is the same thought process. One thing we emphasize is learning Good Laboratory Practices in my group; it gives students a chance to practice and learn how to do these things so they can apply them to other applications when they graduate.

Q: What do you enjoy most about your work?

A: Personally, I enjoy working with the students, training them, and exploring what the possibilities are for their work. When they’re learning these new techniques, the students often find there are lots of ways we can go, and we’re always coming up with new ideas and trying to pursue those ideas. It has been fun trying to see exactly what we can do with HPLC that people haven’t done before. I think there’s a lot of potential there in characterizing biological systems that hasn’t really been done much in the past. There is also still a lot of potential for developing more selective methods by combining different chromatographic methods. This approach has been used before, but I think there’s a lot more room for growth there as well.