Lessons Learnt from HPLC

High performance liquid chromatography (HPLC) has become an essential tool for the advancement of biomedical research. During the past 25 years that I have been using HPLC, there have been enormous advances made in areas such as column technology, solvent degassing, pump pressures, and gradient dwell volumes that have all led to substantial increases in the robustness of the instruments. My career has aligned principally with the rise of LC-MS. I remember spending countless hours trying to set up thermospray and flow-fast atom bombardment experiments. In those days, we just hoped we might get one – or if we were really lucky – two LC-MS runs accomplished in a day. The routine use of LC-MS that we see today was almost unimaginable back in the 1980s.

I still remember my first exposure to HPLC as a summer intern in drug metabolism for a pharmaceutical company. I was asked to make a fluorescent derivative of an analyte to improve its response. It was a simple Michael addition reaction involving N-pyrene maleimide with a sulfhydryl group. It was a simple enough reaction but as I optimized the chromatography I began to resolve my derivative into two peaks. I remember going to my supervisor and discussing my results and us both scratching our heads. We finally built a model of the derivative and realized that depending on how the derivative formed, through attack from above or below on the sulfhydryl group, it would result in two different isomers. I have always remembered this experiment as an excellent example where the existence of extra peaks in a chromatogram led us to a deeper understanding of the chemistry that was being used.

There is one other important lesson that I learned that summer that I have always kept with me and repeated many times to my students. I was developing a method for a compound and based on the pKa needed a lower pH buffer than I had been used to using. One of my colleagues told me to look at several organic acids and then go see what we had available on the shelf. I settled on a citrate buffer and ran my first sample and had a very nice looking peak. I then ran the sample again but the retention time decreased a little bit. I thought that was odd and ran it again and the peak again came out earlier. At this point I went to seek advice. After several people confirmed that was odd and agreed that the retention time kept getting shorter and shorter with each successive injection someone finally asked to look at the information sheet that came with the column. After a few minutes they handed it back to me and said, “There it is.” I was surprised when I read point #17 on the technical sheet for the column which said, “Do not use citrate with this column.” Our head chromatographer told me not to worry because that was really odd and he had never seen that before, but unfortunately I was slowly removing the stationary phase from the column with my buffer.

While over the years I have certainly had many far more expensive lessons, you always remember your first one. Therefore to this day, I never use a column without scanning over the little leaflet that comes with it just to make sure there isn’t some strange warning there that I wish I had known.

Michael G. Bartlett received his B.S. in chemistry from North Carolina State University, Raleigh, NC, USA, in 1990 and his Ph.D. in analytical chemistry from Georgia Institute of Technology, Atlanta, GA, USA, in 1994. Currently, he is Professor at the University of Georgia, Athens, GA, USA, and Interim Assistant Dean for Nontraditional Education and Outreach.

Mike is currently Editor-in-Chief of the international journal Biomedical Chromatography.