Bioanalysis would be much different without LC-MS. And ever since I was a youth I have been fascinated by history. There are a few words of history that I won’t forget. “Those who fail to learn history are doomed to repeat it” My favorite history as related to science quote goes along the lines of “nothing new is ever invented/nothing new under the sun”. Authors of these quotes are unknown. Connecting a high vacuum mass spectrometer with a liquid separation technique wasn’t straight forward and eluded researchers for decades. Before we talk about the “MS Tail that Wagged the HPLC Dog” (ESI-MS Nobel Prize winner John Fenn Quote), we must discuss the history of MS and HPLC separately, as the divergence and convergence is important. I also must give credit to my Ph.D. advisor, Dr. Phyllis R Brown – a historian, excellent author and leading scientist for HPLC in the 1960s until her retirement in 2000s. – she so much enjoyed telling her stories to her students about the early research in HPLC.
Runge, Schonbein et. al. described separations of solutes on paper in the 1850s and 1860s. The history of column liquid chromatography starts with Dr. Mikhail Tsvet. Dr. Tsvet was a Russian Botanist who wanted to separate the pigments in a leaf. Thus in 1900 using petroleum ether as the “mobile” phase and calcium carbonate packed into a glass column as the “stationary” phase, he separated the chlorophylls in a leave and could visually see orange and green colors separate through the process. He called this “chromatography”. In Russian his name means color. Chromatography means “color writing”. That is oddly coincidental, ironic or is it premonition? The work was described in 1901 and wasn’t documented in writing until 1906. Sadly, with all the upheaval in Russia at the time, his work was lost for decades. He was nominated for the Nobel Prize in Chemistry in 1918 (no prize awarded that year) and passed away in 1919. A note to parents, reproducing this work for Science Fairs for children obtains great rewards!
In 1941, Martin and Synge developed the next seminal piece of work with the development of liquid-liquid chromatography. The researchers used silica gel to “hold” water and then an organic solvent was passed through the column to separate amino acids. Martin and Synge received the Nobel Prize in Chemistry for this work in 1952. This work further led Martin to the invention of gas chromatography in 1949. According to my Ph.D. advisor, Dr. Phyllis Brown, Martin and Synge actually stated, “…this technique we describe will never be successfully applied on a large scale as it is a serial technique”. Well, they were right – the bottle neck of much research is “one injection/detection at a time”, whereas other research can be done in parallel. Researchers continue to try to do analytical techniques in parallel and it continues to be elusive in a practical setting. This is why speed in bioanalysis is so important.
J Calvin Giddings described in the 1960s, the potential to drastically reduce the current particle size of packings from 150 μm to below 10 μm and use pressure to push the mobile phase through the column. His theory was correct and led to more efficient separations. He even wrote a book titled, “Dynamics of Chromatography: Part 1”. He was nominated for the Nobel Prize in 1984 and 1994. Csaba Horvath is often thought of as the first researcher to use high “pressure” liquid chromatography while a Professor of Engineering at Yale. Many think he should have won the Nobel Prize. I was honored to be an invited speaker at his celebration of life session in 2004 – world renowned chromatographers were in that room including my advisor Dr. Phyllis Brown. I met so many great scientists that day.
A few stories from Dr. Phyllis Brown re: Calvin and Csaba. Dr. Brown asked Calvin, “You wrote, Dynamics of Chromatography Part 1, where is part 2?”. He replied, “I never wrote it”. According to Dr. Brown, Csaba was an analytical chemist to the millionth degree but Yale and all Ivy League Schools in the 1960’s cancelled all their Analytical Science Chemistry programs as they didn’t it was a “real” science discipline. It is why Dr. Brown left Brown University as a post doc and went to University of Rhode Island. It is also why the Midwest and schools such as Michigan, Purdue, and UNC have great analytical programs – the Northeast didn’t want them.
Another story from Dr. Brown was how Calvin Giddings came up with the term, “eddy diffusion” in chromatography. Calvin was an avid kayaker and he noticed in rivers along the edges, that the flow of the river “swirled” and even “went in reverse”. This is called an eddy in a river and it happens inside a chromatography column near the column wall and causes irregular flow, diffusion and thus broader peaks. Thank you Dr. Giddings for kayaking and the term “eddy diffusion”!
HPLC wouldn’t be complete without mentions of Jack Kirkland, Richard Henry and Ron Majors. They all were major influencers in HPLC packings and are all somewhat connected to Dupont. I had the honor of meeting all of these researchers including teaching LC-MS courses with them and designing stationary phases with them for LC-MS. Jack is considered one of the original researchers to bond a liquid phase to small silica particles (sub 50 μm) that led to “Zipax” and “Zorbax” and likely the first to bond a hydrocarbon to silica (reversed phase). Ron Majors did crucial research in the area of sub 10 μm particles for high efficiency separations. Much of the research by Jack Kirkland previously was using gas chromatography and being a member of the Biochemical Division at Dupont, Jack soon realized the limitations of GC for biomolecules due to their non volatility. Without these scientists, bioanalysis as we know it today, wouldn’t exist.
I am proud to say that Dr. Phyllis Brown was THE or one of THE first researchers to apply “biomolecules” to HPLC in the 1960s. At Brown University during her Post Doc she used a “DNA Analyzer” called that by the manufacturer. She would chuckle when telling her students, “It was just an HPLC”. She analyzed nucleotides and nucleosides using HPLC and her research into separations and analysis of those molecules continued until her retirement. Without her research, analysis of oligonucleotides wouldn’t be possible – an important part of bioanalysis today. And my own research with her, “Design of HPLC Stationary Phases for LC-MS, wouldn’t have been possible.
In the 1960s and 1970s, manufacturers such as Waters and E. Merck were important in the development of silica HPLC particles such as Corasil and Perisorb, respectively, with particles in the sub 10 μm range. In the continued search for the most efficient separation, particles continued to decrease in size including sub 2 um particles in the 1990s and 2000s by several manufacturers and porous layer particles, which give improved efficiency without the increase in back pressure.
I love history and I could make this blog an entire book to discuss, Van Deemter (A, B, C terms) reverse phase, normal phase, HLIC, ion-exchange, affinity chromatography, size exclusion, capillary electrophoresis, HPLC on a chip, GC on a chip, capillary electrophoresis on a chip, and I will leave this for another time. The important part is to review the history of HPLC and realize without these scientists, bioanalysis wouldn’t be possible. I also realize how fortunate I was to meet so many of these scientists face to face and have an amazing research advisor.
I want to give a shout out to all the scientists who have contributed to HPLC and separation science. Without their contributions, Veloxity Labs wouldn’t exist and “treating disease one sample at a time” would have an entirely different meaning.
Contact us to discuss your next project where using HPLC AND MS we continue to “MOVE AT THE SPEED OF BIOANALYSIS”.
Shane Needham, Ph.D.
Co-Founder, CEO and President
Veloxity Labs, LLC
