The first 50 years of HPLC—A Parallel Journey

ORIGINAL 

With its beginning in 1966, HPLC is entering its 50^th year, celebrating its Golden Jubilee, and now is the single biggest chromatography technique indispensable to most laboratories of the world in helping to solve “real world” problems (1).

The development of HPLC parallels the development of my own career. I was a graduate student at Purdue University working on my Ph.D. in analytical chemistry (1963-1968) when the research into what eventually became HPLC was just beginning. Since my doctoral research was in the area of silica chemistry, I was closely following the work of Lloyd Snyder, Ralph Iler and J.J. Kirkland at Dupont and other workers who eventually became the “founders” of HPLC. My thesis research dealt with what today is known as molecular imprinted polymers (MIPs) but then we called them tailored adsorbents. Although most measurements on my research involved the development of adsorption isotherms, working in the laboratory of Professor L.B. “Buck” Rogers, a legend in separation science and other analytical fields, with fellow students performing classical low pressure LC and GC, I couldn’t help being exposed to gas- and liquid-chromatography every day. In fact, before getting my degree, Brian Bidlingmeyer, who I overlapped with for two years at Purdue, in the late ‘60s undertook the first project involving UHPLC (Ultrahigh Pressure Chromatography) performing chromatography at 3500 kg/cm² (~50,000 psi) (2,3). Because Buck was extremely cautious about using such high pressures around the rest of us students, he relegated Brian to a laboratory way down in the basement of the chemistry building in case a column would explode under the high pressure. Brian’s columns resembled rifle barrels.

            While in graduate school, I had the opportunity to learn how to write scientific reports and papers. Buck expected each member of our research group to write a dreaded weekly report on our progress since last week’s report. Most of us felt that it was a big pain to write a report so often especially during the hot, muggy summer months in West Lafayette, IN. The chemistry building had no air conditioning so we spent a lot of our summer hours at the co-recreational swimming pool or at the Pub, our favorite drinking establishment. Not much time to do research except at night when things cooled off a bit. Buck’s reasoning about the reports was that once you get out into industry or academia, you will be writing many reports or grant proposals, like it or not. In retrospect he was right and to this day, I have not found technical writing to be a chore. Buck also always encouraged his students to give oral papers and during Pittcon 1964, I gave my first public presentation on my research. Believe it or not, Pittcon was still being held in Pittsburgh but there was no convention center and no hotel could hold this growing conference which needed lots of meeting and exhibition space. For many years, Pittcon was held in the historic William Penn Hotel in downtown. The actual exhibition was held in individual hotel rooms on the various floors. Each exhibitor had a hotel sleeping room where their goods were on display and an attendee had to traverse multiple floors to see the product displays. After that, Pittcon moved to the Cleveland Convention Center and has never returned to Pittsburgh even though there is a lovely and large Convention Center in the Golden Triangle.

The job market in 1968, the year that I received my Ph.D., was great and never having the opportunity to travel, I interviewed every company that came by Purdue looking for analytical chemists so I had 13 trips around the U.S. Since Buck was a Dupont consultant, everybody from Buck’s research group got invitations to three Divisions of Dupont in or near Wilmington, DE. Lo and behold, I had a chance to visit the lab of Iler and Kirkland at the Industrial and Biochemicals Division (no longer exists) where I had a chance to meet some of the best silica chemists in the world. I was in awe. A visit to Union Oil in Brea, CA, resulted in a similar occurrence with another one of my heroes. Lloyd Snyder was my host and so another day of silica discussions and interviews. I was pleased when Lloyd asked me to dinner at his home.

Normally, when I was interviewing for a chemist job for after grad school, my host would take me out to a fancy restaurant and we would talk about things other than science. In this case, Lloyd invited me to his house for a home cooked meal prepared by his wife Barbara. It was a very nice gesture and I thought it would be a relaxing evening after a full day of interviewing, which is always full of “tension” for the prospective employee. But, alas, we ended up talking about chromatography and chemistry the whole night, which I enjoyed but felt that the interview was continuing at his home. All in all, if I had taken the job at Union Oil, I wouldn’t have been on the East Coast and wouldn’t have met my wife, Carol, a New Yorker, so it all worked out in the end.

My First Job and An Exciting Symposium

Although I was hoping to get back to California, my best job prospect, in 1968, despite all the job interviews, was at the Celanese Research Company in Summit, NJ where I was to head up the separations laboratory. Not only did I get a very responsible job, but I had some technicians well versed in gas chromatography who could carry out the daily work supporting researchers in a variety of application areas. However, there was nothing in the way of a liquid chromatograph in the lab or that one could purchase since HPLC as an instrumental technique was non-existent. Since many of the samples encountered in this Celanese central research lab were non-volatile and not amenable to GC, I felt that this was an opportunity to explore this new technique that had no defined name at that time. I talked to my boss to let me attend one of the Advances in Chromatography Symposia, a series started by Prof. Al Zlatkis of the University of Houston. The 5^th meeting in this series was held in Las Vegas, NV in January, 1969. The previous four were devoted to GC, an area of focus for Zlatkis. Naturally, the largest contingent of the attendees and the presentations were devoted to GC which by now was a very established separation technique. However, I was there mainly to hear about new liquid phase possibilities. Les Ettre, in one of his Milestones in Chromatography articles (4) made a nice presentation on this historical meeting but never captured the state of excitement that existed in the LC sessions at that time, especially for a young Ph.D. starting his career who wanted to make a statement to prove his value to his company!

Many of the presenters in the LC sessions were notable gas chromatography experts who decided to switch their allegiance to work in this new area that was wasn’t even in its infancy but was still in the birth canal. Names like J.F.K. Huber, J.C. Giddings, R.P.W. Scott, Cs.Horvath, L.R. Snyder, J.J.Kirkland, I. Halasz, and V. Pretorius all now associated as being the “fathers” of HPLC gave their presentations to a modest audience of listeners including yours truly. The really exciting part came when the discussion session on LC was held. Nowadays, such discussions no longer held at international chromatography meetings probably because of the vast venues don’t lend themselves to intimate face-to-face discussions. Also, back in the early days, people weren’t as politically correct as they are now and when researchers had conflicting views on the theory, practice and implementation of LC, heated arguments took place right on the floor. The entire proceedings of the Las Vegas meeting were published by Preston Technical Publications. I found my copy on my bookshelf and was surprised to find out that it still had my original folded notes folded inside!

My First Liquid Chromatograph

So, when I got back to my Celanese lab, I decided that I could build my own LC instrument (6), as many others at the time did also. High-pressure pumps were available but not built with LC in mind but more for process chemistry so these pumps had to be adapted. For a pulsating pump, a pulse damper was needed. Fortunately, Waters had one for their GPC unit that was sold separately and worked to 2500 psi; above that I had to use an accumulator which was a device charged with gas under pressure and when installed in a high pressure flow line would absorb pump pulses. Other components were added as needed such as a pressure relief valve for safety, a pressure gauge, a pre-saturator column for liquid-liquid chromatography operation, and a flow splitter for filling the reference side of a flow-through refractive index detector. The other detector I used was a Laboratory Data Control UV detector operating at 254-nm and was the newest piece of equipment on the block. The biggest challenge was to find an injector that would operate at pressures up to 5000 psi. Valco and Rheodyne weren’t making 6-port valve injectors for high pressure liquid so I had to build my own. Fortunately, C.D. Scott of the Oak Ridge National Laboratories, who was a pioneer in ion exchange chromatography, published an article in Analytical Biochemistry (5) where he gave directions on building such a valve. His valve was way too big and, to cut down on dead volume, every channel had to be reduced by a factor of 100. I had to make my own endfittings by sawing a Swagelok union in half and then have our machine shop cut a capillary union in half and weld the two together. The system (Figure 1) was for isocratic work only (isocratic wasn’t even a word then but was later coined by Horvath and gradient elution was called solvent programming) and had no temperature control. Getting started in HPLC/UHPLC is much easier nowadays.

Column selection was pretty straightforward. Eveybody, including me, was enamored with the latest superficially porous particles: Zipax (20-37µm, controlled surface porosity support from Dupont for liquid-liquid partition chromatography); Corasil I and II (37-50 µm, porous layer silica from Waters, best for adsorption chromatography); and the only bonded phase product available, Durapak-OPN (which was actually a totally porous bead of 36-75 µm with –OCH₂CH₂CN “brushes” extending from the surface, also from Waters). The later was unsuitable for use in any solvent containing water which would shave the “brushes” off of the packing material (cleave the Si-O-C bonds). Reversed-phase chromatography columns weren’t a reality yet. Because of their large particle sizes, all of these packings could be dry packed into stainless steel columns 1000-mm in length and 2.1-mm i.d. which was the standard size of columns used with these LC supports.

Since I was working for an industrial company that had a need for applications, I worked on a problem that we had of analyzing antioxidants and plasticizers in polyacetal and polyethylene samples. After ether Soxhlet extraction of finely ground polymer samples, I was able to determine and quantitate trace amounts of three hindered phenol antioxidants: butylated hydroxy toluene (BHT), CAO and Santonox R in each polymer (6) all in a matter of 5 minutes. I presented my work at the next Zlatkis 6^th Advances in Chromatography Symposium in Miami, FL, one year after I set out to build my own chromatograph and get a valid application. In retrospect, I found that this paper was one of the first applications of HPLC to solve an actual industrial problem and not just running a bunch of standard compounds.

Back to California and Early Contributions

Coming from California, I kept my eyes open for the opportunity to return to the Golden State. After I had published my work and gave some lectures on HPLC at various meetings, Bob Stevenson, who was an applications chemist at Varian in Walnut Creek, CA, mentioned to me at an Eastern Analytical Symposium meeting in the Fall of 1970 that Varian was looking for an R/D chemist for their liquid chromatography program. Although I was relatively happy at Celanese, I jumped at the chance to get back to the West Coast. I interviewed for the job, got an offer, accepted it, married my girlfriend and left with my new bride for California all in a span of three weeks. Starting work in February, 1971, I ended up spending the next 17 years with the organization.

Varian Aerograph was well established as a player in the GC market and was interested in getting their feet wet in this new area of LC. At the time, Varian had taken over the Picker Nuclear Nucleic Analyzer system developed in conjunction with Professors Csaba Horvath and Sandy Lipsky of Yale University (7). In reality, this was the first true HPLC instrument on the market but was in need of an overhaul to make it more applicable to general purpose HPLC. In addition, the two professors had spun off a small company called Northgate Laboratories which was based on their research work on ion exchange pellicular packed microbore columns. My technician, Margaret Hawken, was a real expert at keeping the LCS-1000 (as it was designated at the time) running and keeping the columns from falling apart. The columns were 3-m long by 1.0 –mm i.d. and were wound in a coil much like a GC column. Unfortunately, the biggest problem that we encountered was the coated polymeric layer flaking off the solid glass bead and plugging the tiny frit at the outlet of the column. We had a regular flow of columns from Connecticut, home to Horvath and Lipsky, to California while trying to solve this problem. The instrument was even capable of running gradients which was needed to elute nucleotides and nucleosides from biological extracts.

During this time, we also had a chance to work with Phyllis Brown who was a researcher at Brown University and was one of the few academics and females working HPLC, especially in the life sciences (8). She also had a loaner Nucleic Analyzer system so we could compare notes. Dennis Gere of Varian, who was working on the East Coast at the time, was a frequent visitor to Phyllis’ lab to trade war stories. She was a pleasure to work with and I will always admire her courage and perseverance in dealing with the old boy’s network! In the end, she was recognized by her peers as a real contributor and innovator in the application of HPLC to “real world” biological problems.

Since I was hired at Varian to work on columns especially for an upcoming syringe pump system that was supposed to blow all other early HPLC systems away, I began working, like everybody else, on the superficially porous packings. These were great because they could be packed like a GC column with a funnel and a vibrator, had low pressure drops because of their relatively large particle size and, once you got the hang of working with liquid-liquid chromatography, problems could be solved and chromatograms generated. Still, everything wasn’t rosy. The SPP packings did give some separation but their sample capacity was low, the silica beads gave tailing, and the efficiency had to be improved-remember the prediction of Martin and Synge in 1941 (9) that clearly stated that “…the smallest HETP (Height Equivalent to a Theoretical Plate) should be obtainable by using very small particles and a high pressure difference across the length of the column”.

Sometime in 1971, Varian Chromatography Marketing Manager, Paul Batchelder, received a bottle of a new silica material from Brinkmann Instruments, who at that time was a dealer for E. Merck, Darmstadt, Germany (now called in the United States EMD Millipore). He handed me this bottle which I still have as a memento (Figure 2) and asked me to see if I could pack this silica into an LC column. Well, I told him that I would get to it eventually and put it on the shelf. Every day this little yellow bottle would stare at me and I kept procrastinating about Paul’s wish. It turns out that inside this bottle was 10 grams of a narrow distribution (only 5-10 µm) of Merck’s TLC irregular EM 50 silica gel that had been sized by air classification. Since there wasn’t much silica gel to play with and having tried to dry pack sub-20 µm silica earlier, I was pretty sure that there had to be another way. Professor J.F.K. Huber of the University of Amsterdam, The Netherlands had gotten a kieselguhr sample (5- to 15-µm) and was trying to dry-pack these particles using a technique that he had perfected for larger particles that involved careful hand packing and tamping each portion of silica using a PTFE-tipped rod (10). He was able to get highly efficient columns but because the particles weren’t densely packed, the column lifetime was extremely short. Not an ideal situation.

So, I started to look into various slurry packing techniques. A slurry would allow the particles to be pumped into an empty column and since no drying packing was involved presumably the column would be more stable. After trying different slurry solvents with some success, I came upon a technique that had been used for larger size-exclusion particles called a balanced- density slurry method (11). The basic idea was that one would suspend the silica particles in a solvent which would have an equal density to the silica so that during the packing procedure, the particles would not segregate by size and thereby form a homogenous packed bed. After trying different ratios of dense, nasty solvents tetrabromoethane and tetrachloroethylene, I came upon a solvent mixture that seemed to be ideal. The method was found to produce very efficient columns that were stable for many injections. Shortly after completing this work, on a trip to Amsterdam, The Netherlands, I had the occasion to show Prof. Huber some of my data. He was thoroughly impressed and told me so which made me feel great coming from such an icon. A publication in Analytical Chemistry resulted (12) and Varian became the first company to have a high efficiency, high capacity microparticulate column on the market, called MicroPak. I felt that this project was one of my most successful endeavors.

Over the next few years, the superficially porous particles slowly died out and the microparticulates took over in most applications. Shortly after MicroPak columns were introduced, Dupont came out with Zorbax, a spherical microparticulate silica material, developed by Kirkland (13) and produced by Agilent Technologies even today. Spherical microparticles packed more uniformly than irregular silica particles and are today’s mainstay column packing base material for HPLC/UHPLC.

This initial study was followed up with a more thorough study on the effect of particle size on column efficiency where a series of different particle sizes and distributions were evaluated and compared to superficially porous particles (14). The van Deemter plots in this paper showed that narrow distribution of porous particles of an average diameter 30 µm when slurry packed was equivalent to superficially porous particles (SPP) with an average diameter of 43 µm (Figure 3). Slurry-packed porous particles below 30 µm outperformed SPP of any available particle size. For dry packed porous particles and slurry packed porous particles of the same average diameter, the slurry packed columns of consistently outperformed the dry-packed columns. Slurry packing has been used for the last 40+ years for all commercial analytical columns.

 In 1973, HPLC was just starting to catch on and the Zlatkis 8^th Advances in Chromatography meeting moved across the border to Toronto, Canada. Still dominated by GC presentations (80% of the lectures), HPLC was finding its place and topics like gradient elution (still called solvent programming), siloxane chemically bonded phases (now taking foothold), and a few applications were noted. I presented a lecture (15) on techniques for LC columns packed with small porous particles and showed a number of applications on microparticulate C18 and cyano siloxane bonded phases which by now were beginning to blossom as the way to go for serious LC work, for both isocratic and gradient solvent systems. Even SPP bonded phase packings were on their way out.

In 1974, another paper involved a systematic study of chemically bonded polar and nonpolar siloxane phases with single step and multistep bonding procedures (16). In this study, we found that monolayer bonding surpassed polymeric bonding in terms of reproducibility and that longer chain alkylsilanes gave more retention in the reversed phase mode. Some usual selectivity effects were achieved for a series of steroids when comparing polar phase such as cyano, pyridyl, aminopropyl, and isocyanatopropylmethyl. A variety of hydrophobic reversed-phase ligands studied like benzyl, phenyl, C6, C10, C18, vinylphenyl, vinylmethyl, and allylphenyl were studied, several functionalities which are now in commercial columns. Infrared spectroscopy of the bonded silicas was used to following the bonding procedures and estimating the number of unreacted silanols.

Across the Ocean to Another Side of the Chromatography Picture

In early 1973, although I was doing fine in my role as an R/D chemist at Varian, I was presented with the opportunity to go to Europe as a technical specialist. I was warned by my R/D colleagues that this would ruin my scientific career but I was still young, my wife and I had no children, and I was interested to learn about other cultures and see some of Europe. So I agreed to a three year temporary stint. Varian’s European headquarters was in Zug, Switzerland but the highly nationalistic Swiss wouldn’t issue me a work permit at my lowly level so we lived in England during my time there. For the next years, I spent at least one week out of every month in Zug. My assignment was to help launch Varian’s entry into the HPLC instrument business with their own developed product, a high pressure syringe pump (Model 8500) that could achieve pressures up to 5000 psi. At that time in HPLC, there were three types of pumps: 1) reciprocating pumps; 2) constant pressure pumps and 3) syringe pumps. Reciprocating pumps are still the most popular today but in 1970s the jury was still out. A big advantage of syringe pumps is that they were very precise on delivering solvent since they were driven by a screw driven motor and could go to very high pressures and, unlike the other two types of pumps, had no pulses,. A gradient could be formed by having two pumps and teeing them together (see Figure 4). The downside of the syringe pump was when the solvent chamber was empty, the pump had to be refilled but that could be figured out ahead of time so it wasn’t a big problem in its applications.

 Things were moving along fine until we found out that there was a potential problem with syringe pumps when used in the gradient mode. It all had to do with bulk solvent compressibility. As one comes up to pressure with a syringe pump, the solvent is compressed, not as much as a gas, but slightly. This compressibility was not a major problem when the pump was used in the isocratic mode and the syringe pump was designed to come up to pressure very rapidly with a valve that was closed during pressurization. When two pumps had to be refilled this wasn’t a problem either since they were filled independently. However, when the pumps were used in the gradient mode they were connected to each other making sure that the two solvents blended together. An added small mixer ensured good solvent mixing. What happened when the pressure started to change? Each pump kept on pushing at its designated flow rate but the solvent in each cylinder would be compressed to different extents. In simple terms, as the column pressure rose during a gradient the more compressible solvent in one pump would compress more than the less compressible solvent in the second pump and therefore some backflow would occur. Eventually, as the viscosity lowered, the more compressible solvent pump would catchup and some additional compressed solvent would be expelled. All-in-all, this alternating compression-decompression would establish a ringing phenomenon that the user wouldn’t see (unless he spiked one of the solvents with a UV absorber and monitored the gradient) and as long as the pumps started out with the same volume, gradients would be very reproducible.

Unfortunately, Prof. Georges Guiochon and coworkers, being excellent theoreticians, independently discovered this phenomenon from a theoretical basis, and submitted a paper to the 1975 Advances in Chromatography Symposium in Munich, Germany. Knowing in advance that Guiochon was going to focus on the syringe pump compressibility issue, Varian management was up in arms about what to do to “combat” this finding. It was decided to give an opposing lecture at this symposium and present data to show that, although Varian recognized the problem, it did not stop the pump’s performance in practical use. But, there were two problems to face: 1) who would deliver this opposing lecture and 2) since the program was already set how were we going to get the time to deliver the lecture. Problem 1 was easy-I drew the short straw and had to face problem 2 alone. After some negotiation, I got agreement from the Symposium chair to allow me to deliver a 15 minute lecture in the program just after Guiochon delivered his painful (for Varian) lecture. Of course, I was very nervous going up against this chromatography pope but managed to squeeze in my “proof statements” in the allotted timeframe. The surprising thing was that Guiochon, in his lecture, never got to the root of the problem of gradient ringing since he was focused on isocratic use of the syringe pump (17); later they added the gradient case (18,19). Needless to say, the competition pickup on the compressibility problem and, even though some modifications were made to minimize the ringing phenomenon, the syringe pump was doomed forever.

During the 21/2 years that I was there, I traveled all over Europe talking about HPLC systems, columns, detectors and the like. I met many of the European chromatographers and was somewhat active in chromatography groups and meetings,  especially in England. I still have friends today that I keep in touch with from those days. In my time there, I found the average European chromatographer to be more technical than the average U.S. chromatographer. Europeans would question undocumented statements and chromatographic results more than Americans would. Americans were also less resourceful in getting the most out of their HPLC columns and systems, probably because of their “disposable” mentality. For example, Europeans would use column repacking services, recycle solvents and be more likely to pack their own columns. These differences led me to do an LCGC survey comparing U.S. and European column trends (20)

Since I was living in Europe, I got involved with the HPLC series of meetings that began in 1973 in Interlaken, Switzerland. After the 1969 Zlatkis meeting, this was the next pivotal meeting in the history of HPLC. Again, Les Ettre did a very nice job on covering this important kickoff meeting which really set HPLC apart from the masses of other separation science techniques (4). Held on a beautiful mountain retreat where the last several hundred meters was so step it had to be traversed by a cogwheel tram that left one spellbound. The European liquid chromatography leaders put together an excellent program attended by 450 delegates with 55 papers presented ranging from theory, column technology (especially particle discussions and chemically bonded phases), detectors, and eleven applications. I gave a paper (21) co-authored with my Varian boss, Fred MacDonald, comparing microparticulate columns to the superficially porous particles and was the only “theoretical” paper I every published (it had equations). In 1973, HPLC instrumentation were still rather rudimentary and not much attention was paid to this aspect of LC since the Interlaken meeting was populated by mostly academic lectures.

            I got further involved with the HPLC series. I was asked about taking on one of the future meetings. After making a pitch to the Permanent Scientific Committee in 1983, I was granted chairmanship of HPLC 1986, the 10^th International Symposium of Advances in HPLC to be held in San Francisco, CA. I didn’t do it all alone; I had help from the local Bay Area Chromatography Colloquium (BACC) which was a loose conglomeration of chromatographers, mostly from columns and instrument companies, Varian gave me some time to devote to the Symposium and my Secretary, Sally Bird, was a great help in keeping information flowing. Unfortunately, at that time, there was no seed money or any financing available for upfront expenses. Also, remember, in 1984 when I started working on the Symposium, there was no e-mail, no websites, and all the kind of stuff we now take for granted. But one thing I had going for me was that everybody loves to come to San Francisco. My first job was to get a Symposium manager, someone who had done it before. In those days, Shirley Schlessinger was the “go-to” person for organizing these sorts of meetings. She had successful organized the International Symposium on Proteins, Peptides and Polynucleotides (ISPPP) so had some experience in our industry. Shirley was a tough negotiator and was able to get the Sheraton Hotel on Market Street that was about to undergo restoration at a cheap price, especially for San Francisco. There was no convention center that would hold a “smaller” meeting like ours so we took advantage of the situation and got the hotel. But, we had a problem, we still needed money to put a down payment to reserve the hotel and other upfront expenses. So, wearing my marketing hat, I began to hit up the instrument companies who would stand the most to gain by having a successful Symposium. I asked and got 35 companies to pay $500 each as a down payment for their booth two years in advance. Those that did so got a reduction in the final price of their booth and other perks. Now, we had enough for the hotel deposit and to start a mailing campaign and printing flyers, etc. The rest was lots of work with lots of fun in between. Even now 30 years later, I would like to thank my local Committee and the remainder of the Permanent Scientific Committee, a number of whom we have lost over the years, for their help and support.

The final outcome was a very successful HPLC 86 Symposium with 1555 attendees, still the best final attendance of this 43 meeting HPLC series. Shirley had a great payday and there was $175,000 profit that was used turned over to the BACC which eventually became into California Separation Science Society (CASSS) a few years later.

In 1998, I was honored to be asked to become the poster chairman for the HPLC series and kept this “job” for 10 years. It was a real pleasure seeing quality posters, organizing the sessions, presenting the poster awards mainly to students, and working with the poster committee up to 60 members at some of the Symposia that always freely volunteered their personal time to evaluate huge numbers of posters. The largest poster display ever occurred at HPLC 2007 in Gent, Belgium where a whopping 800 posters were shown. Still our Committee tackled these posters with a vengeance and we were still able to find the top 3 by the last day of the meeting. I have always tried to call attention of the poster winners including photos and poster summaries in my HPLC series reviews, usually in LCGC’s September issue. Pictured in Figure 5 is my presentation of a Best Poster award to Isabelle François at the HPLC 2007 Symposium, then a Ph.D. student at the University of Ghent, Gent, Belgium) and now as a business development manager for Waters (as somebody asked, no she wasn’t trying to kiss me during the 2007 award presentation). I do believe that over the 10 year span, because of the increased emphasis on the importance of the posters and the fact that they were given as much credence as orals and were treated as an important part of the Symposium that the quality of the posters improved. Likewise, the Horvath lecture winners for the best lecture by a student presenter also got coverage in LCGC’s yearly reviews of the HPLC Symposia.

Back to the US and More Sample Prep

In 1976, I returned to the U.S. with a new job with Varian in a new location, Palo Alto, CA. I was now more involved in management of the separation science applications laboratory so spent most of my time in meetings and strategy sessions. However, in 1977, after the demise of the syringe pump, a decision was made to get all the expertise in HPLC back to Walnut Creek and have a “crash” project to develop a new concept and get Varian back into the HPLC race. Working on a team where mechanical engineers, electrical engineers, programmers, chemists, technical manual writers and marketing people are sitting together with open partitions really was a rewarding experience. It really emphasized the concept of teamwork and how miraculous things can get done given the right motivation. The result was the first microprocessor-controlled integrated LC system with a CRT and a single pump with low pressure proportioning to form binary and later ternary gradients. After the launch in 1978, Varian leaped back into the market tripling their market share in 18 months, albeit a still small share compared to Waters and Hewlett-Packard. I elected to go on the roadshow team introducing the product and we drove thousands of miles with the instrument in tow to give seminars in a different city every day. It was grueling but fun. Figure 6 shows a celebratory photo which has the 1000^th Varian LC 5000 being shipped and the management team supporting it.

In 1987, Varian acquired Analytichem International, a small company in Southern California that had established themselves in the sample preparation market, especially in solid-phase extraction where they were a leader. Some of my focus was directed away from columns and more towards sample preparation, one of the last frontiers to be tackled in the analytical cycle. Many analysts were still using age-old techniques that were slow, time consuming, labor intensive and is need of more attention particularly in the automation area. One of the first things we did was to sure-up the AASP, Analytichem’s Automated Sample Preparation machine that performed SPE on a 10 cartridge cassette. The instrument was mainly developed by chemists rather than engineers and so there were areas where re-engineering was warranted. Again it stressed to me, the importance of having the right expertise in place to develop a reliable instrument product. But my zest for sample preparation challenges started here (Figure 7).

Back East and a Consumables and Sample Preparation Focus

Once in a while, an opportunity comes along that is too good to pass up. That happened to me in 1987 when EM Science, the U.S. affiliate of E. Merck, Darmstadt, wanted me to come to New Jersey to be the general manager of their chromatography business. After some interviews by the German brass with many personal questions that would never pass mustard in the U.S., I was offered a very lucrative salary and a relocation package to match. So I packed up my family and our California home and moved. Unfortunately, a week after we purchased our brand new house, a lightning strike caused it to burn to the ground but that is a story I’d like to forget. The business of mostly selling chemicals, LC columns, TLC plates, SPE cartridges and bulk packings through distributors was somewhat foreign to me and was very different than direct selling to end users by Varian. However, I learned a lot about TLC, silica gel, sample prep and specialty HPLC columns. Furthermore, I enjoyed my time working with the Germans from Merck on new products and with Hitachi on LC instrumentation but my heart was more tuned to dealing with end users and especially with sample preparation. So, after a couple of years, when Hewlett-Packard had an opening in their Avondale, PA in their Automated Chemical Systems group working on techniques like supercritical fluid extraction, robotics, and autosampler-based sample prep, I jumped at the chance to return to the mainstream instrument business. Starting in 1990, I remained there until my retirement from Agilent Technologies in 2013 putting in nearly 24 years. Interestingly, during my last years, Agilent acquired Varian and I was somewhat reinstated with my old company, totaling 41 years between the two!

During my stay at Agilent, besides sample preparation, I had the opportunity to return to the area of my early interest, LC and GC columns. As a senior scientist, I had a chance to work with the consumables’ team on microparticulate LC/GC columns, SPP columns, SPE, and a wide variety of related products. In my last 10 years at Agilent, I had the informal title of Worldwide Ambassador for the columns and sample preparation areas which allowed me to follow my other passion-- travel to new places, meeting customers, giving lectures, organizing technical sessions, and giving short courses and trainings. In all, I had the opportunity to visit 88 different countries (and not just sitting in an airport transferring planes). But at 72 years old, it was time to turn the reigns over to the younger generation. So I retired in 2013 and am still “sort of” retired today.

Technical Writing and LCGC Magazine

This brings me to another area of focus and that is keeping the world of chromatographers up on the latest technology advances. My first writings about commercial LC columns appeared in American Laboratory in May, 1972 (22) just as the new breed of superficially porous- and microparticulate-columns were getting introduced. There I started to tabulate specifications, descriptions and references highlight products the end users could purchase. I tried to give would-be liquid chromatographers a little idea on how they might go about choosing the best column for their application. All that was really available at the time were larger porous silicas, aluminas, diatomaceous earths and ion exchange particles all above 20 µm in diameter. Superficially porous particles of silica-, alumina-, coated-, and pellicular ion exchange particles were also around. The silica and alumina could be used for liquid-solid chromatography or coated with a liquid phase and be used in LLC. Zipax from Dupont was mainly for LLC. Fortunately, for biochemists there were actually some porous ion exchange resins under 10 µm that had been perfected for amino acid analyzers.

There were also some interesting coated Zipax SPP particles called controlled surface porous particles. Coatings such as hydrocarbon polymer and polyamide were available but users were warned that the phase could strip off. The most advanced SPP were Dupont’s Permaphase ODS (octadecylsilane, C18) and Permaphase-ETH ( a silicone ether with intermediate polarity), and Corasil ODS. All three of these packings had siloxane bonded phase that could be used with gradients, higher temperatures, etc. without fear of stripping the phase. The other aspect of these bonded phases is that the technique of reversed-phase LC (a term also coined by Csaba Horvath who had a knack for clever new names) could be performed. In the pursuing years, the latter technique literally exploded and is still the number one mode today.

I updated my American Laboratory column review again in 1975 (23). So many new columns had been introduced my article took up 16 pages with massive tables of new packings and columns with discussions on packing techniques, how to use columns in gradient elution, and lots of practical information. Word was getting around that I was the “go-to-guy” to write about and organize specialists to write about columns for this new technique. In 1977, the co-editors, Roy Keller and John Q. Walker, of the Journal of Chromatographic Science, one of the major journals of the day for publishing articles dealing with chromatography, asked me to put together an entire monthly issue of September on the state-of-the-art in HPLC columns and column technology (24). I was able to talk experts of the time-including John Knox, U. Edinburgh (theory); Dennis Saunders, Union Oil (LSC); Heinz Englehardt,U. Saarbruecken (LSC moderators); Geoff Cox, Lab of the Government Chemist, UK (bonded phases); Cs. Horvath and Wayne Melander, Yale Univ. (theory and practice of reversed-phase LC); M. Caude and R. Rosset, Ecole Superieure, France (ion exchange); and several more on GPC, GFC, and biochromatograpy as well as my “update” on commercial columns with a whopping 322 references-to contribute and this special devoted issue went over very well with newcomers to LC.

Now HPLC was a really hot topic. So in 1980, the editors again asked me to put together not one but two monthly issues (September and October) devoted to some of the same issues but with additional “hot” topics of the day including column care and use, column design, columns for water-soluble industrial and biopolymers, ion chromatography, practical aspects of normal phase and ion exchange chromatography, ion-pairing RPC, open tubular and micropacked capillary columns, and preparative chromatograpy (25, 26). In addition to some from the 1977 issue, I was able to corral some additional well-known experts of the ‘80s including: Novotny, U. Indiana; Regnier, Purdue; Barth, Hercules; Rabel, Whatman; R.P.W. Scott, Hoffman La Roche; Ishii, Nagoya Univ; and Pohl, Dionex. The October issue added additional experts including Verzele, U. Gent, Belgium; Bidlingmeyer, Waters; and Cooke, Altex later to become part of Beckman; and a few others.

I had a chance to expound on a favorite subject of mine at the time (and currently still is): multidimensional chromatography. In this technique, a fraction(s) from one chromatographic column is selectively transferred to one or more secondary columns for further separation. It is a technique mainly for multicomponent samples where one desires to isolate analytes of interest from a difficult background sometimes by heart cutting. Compounds of interest are sent to a secondary column while those with no interest are directed to waste. More recently, comprehensive LCXLC (27) has generated interest but in this technique all fractions from the primary column are directed to the secondary column and represents a bigger challenge than heart cutting. But let us return to 1980, at Varian we had been fooling around with the technique in the lab for a few years. We had successfully coupled exclusion chromatography (primary mode) to reversed-phase chromatography (secondary mode) on-line via high pressure valving and we able to apply the technique to study difficult samples such as additives in rubber, pesticides in vegetables and limonin in grapefruit (28).

We had a new challenge-how to couple HPLC with GC on-line, two seemingly incompatible techniques. Off-line LC-GC techniques where workers would collect fractions from the LC, concentrate the sample or totally evaporate the solvent and re-dissolve each fraction of the sample up in a GC-compatible solvent had been done for years. Florsil cleanup of pesticides in food by column chromatography followed by GC analysis of pesticide fractions eluting from the column was a frequently used technique. Stuart Cram, the Varian R/D Manager at the time, and I were in the lab one night and decided to attempt to interface an HPLC system to the Varian GC autosampler and then direct fractions into a Model 3700 gas chromatograph. In its normal configuration, this autosampler was loaded with sample from a vial and a portion measured by a syringe and injected into the GC. From Figure 8, without getting into the details, one may be able to understand the principle of how the two instruments were connected via the flow-through side arm syringe. For details see (29). An application of this LC-GC system of the determination of atrazine, a selective herbicide, in a sorghum extract proved the feasibility of this combination. Other applications included phenols in water, antioxidants in oil, pesticides in butter, and polychlorinated benzenes (30-31). As far as I know, this was the first successful application of on-line, direct interface LC-GC system.

In the early ‘80s, I became the lead author of Analytical Chemistry’s Biannual Reviews on Column Liquid Chromatography. This turned out to be a rather large undertaking given the large number of HPLC papers being generated in the 80s. I took on Howard Barth of Hercules and Charles Lochmueller of Duke University as co-authors which proved to be necessary. We kept this activity until 1988 adding Fred Regnier of Purdue (1986) and Bill Barber also of Hercules (1988) along the way. After four issues, it was time to turn over the Reviews to a new team. Along the way, though, I was able to learn about other aspects of HPLC besides column technology. My other service for Analytical Chemistry was serving on the Instrumentation Advisory Board from 1987-1989.

In early 1983, I received a phone call from Tom Hager, the Editor of a brand new journal called LC-Liquid Chromatography and HPLC Magazine. He asked me if I wanted to write a monthly column. Of course, I couldn’t turn him down-a chance to write on my favorite topics on just about anything I wanted. The first issue, published in March, 1983, was already underway and had no permanent columnists. I wrote my first article for the April, 1983 issue making me the oldest continuous columnist in the publication. For the 25^th Anniversary Special Issue, I wrote a lengthy (as usual) article entitled “25 Years of Technical Writing for LCGC-My Personal Reflections” where I covered the early days of the magazine and a lot of the extra activities that I undertook for the magazine. I will not repeat all of that material here since it can be found in the original publication (32) and is archived on the LCGC website:

http://www.chromatographyonline.com/25-years-technical-writing-lcgc-my-personal-reflections.

Just for the record though I thought I would summarize some of the Special Issues that I have edited in order to bring some added value to readers of LCGC. I have tabulated these projects in Table I. These issues, mostly mini-reviews on the state-of-the-art in LC column technology and modern sample preparation, allowed us to explore more in-depth on research efforts, applications, and the more practical considerations of separation science. I am indebted to those authors who helped to add to the reader’s knowledge with their focused efforts.

Ever since 1995, I have teamed up with co-LCGC columnist John Dolan to provide a Guide to LC Troubleshooting wall chart every couple of years. This chart is updated as new technologies unfold such as when UHPLC troubleshooting tips were added, we included topics more pertinent to that new technique. Most of our troubleshooting tips in the wall chart deal with column and mobile phase problems such as recovery, pressure, retention, baseline, leaks, and efficiency concerns. In addition, I have been assembling a Guide to Sample Preparation wall chart on a regular basis. These wall charts consider modern techniques like QuEChERS. liquid-liquid extraction, solid-liquid extraction, SPE, Soxhlet extraction, SFE and is based on a decision-tree flow chart. Wall charts are shipped with regular issues once or so per year. GC columnist John Hinshaw and I have gotten together and developed The Chromatography and Sample Preparation Technology Guide. This Guide is based on glossaries that John and I developed and have published over a number of years. This latest Guide was published in September, 2013. The booklet is a glossary of common and not-so-common terms used in chromatography and sample prep and was appreciated by neophytes and old timers alike.

Conclusions

In this somewhat rambling trip down the chromatography memory lane, I have tried to depict how elements of my career have paralleled the development of HPLC and UHPLC and to some degree in sample preparation and automation. I have been working in this technology area for 50 years specializing in HPLC, GC, and sample preparation both on the consumables and the instrument sides of the business. In some cases, I was a contributor, in many cases, I have served as an “educator” through my technical writings in various publications including nearly 33 years for LC- and LCGC-Magazine, lectures at local, national and international symposia, trainings around the world, organizing short courses and sessions at various symposia and seminars at Varian, EM Science and HP/Agilent customer’s facilities. I owe a lot to my managers, colleagues, fellow students, chromatography heroes, teachers, readers, editors, guest authors and co-authors and customers who have helped me and educated me along the way. I hope to continue to offer my assistance to LCGC in the future so maybe you haven’t seen the last of me. 

References

1.    REM article in regular Nov. 2015 issue of LCGC

2.    B.A. Bidlingmeyer, R.P. Hooker, C.H. Lochmueller, and L.B. Rogers, Sepr. Sci. 4, 439-446 (1969).

3.    B.A. Bidlingmeyer and L.B. Rogers, Anal. Chem. 43, 1882-1883 (1971).

4.    L. Ettre, LCGC 18(7) 704-714 (2000).

5.    C.D. Scott, W.F. Johnson and V.E. Walker, Anal. Biochem. 32, 182 (1969).

6.    R.E. Majors, J. Chromatogr. Sci. 8, 338-345 (1970).

7.    Cs. Horvath, B.A. Preiss, and S.R. Lipsky, Anal. Chem. 39, 1422 (1967)

8.    P.R. Brown, J. Chromatogr. 52, 257 (1970).

9.    A.J. P. Martin and R. L. M Synge, Biochem. J. 35, 1358–1368 (1941).

10. J.F.K. Huber, Chimia, Suppl. 24 (1970.

11. Waters Associates, Framingham, MA, “Column Packing Procedures for Polystyrene Column Packing”.

12. R.E. Majors, Anal.Chem. 44(11), 1722-1726 (1972).

13. J.J. Kirkland, J. Chromatogr. Sci. 10(10), 593-599 (1972).

14. R.E. Majors, J. Chromatogr. Sci. 11, 88-95 (1973).

15. R.E. Majors, Anal. Chem. 45(4) 755-762 (1973).

16. R.E. Majors and M.J.Hopper, J. Chromatog. Sci. 12, 767-778 (1974).

17. M. Martin, G. Blu, C. Eon and G. Guiochon, J. Chromatogr. 112, 399-414 (1975).

18. M. Martin, G. Guiochon, G. Blu and C. Eon, J. Chromatogr. A, 130 (11) 458-459 (1977).

19. M. Martin and G. Guiochon J. Chromatogr. A, 151 (3), Pages 267-289 (1978)

20. R.E. Majors, LCGC 7(6), 468-475 (1989).

21. R. E. Majors and F.R. MacDonald, J. Chromatogr. 83, 169-179 (1973).

22.  R.E. Majors, Amer. Lab., May, 1972.

23.  R.E. Majors, Amer. Lab., October, 13-36 (1975)

24. R.E. Majors, J. Chromatog. Sci., Special Issue 15(9). 333-440 (1977).

25. R.E. Majors, J. Chromatog. Sci., Special Issue 18(9). 393-486 (1980).

26. R.E. Majors, J. Chromatog. Sci., Special Issue 18(10). 487-582 (1980).

27. P. Schoenmakers, LCGC No. America 26(7), 600-608 (2008).

28. E.L. Johnson, R. Gloor, and R.E. Majors, J. Chromatog.149, 571-585 (1978).

29. R.E. Majors, J. Chromatog. Sci. 18, 571-579 (1980).

30. S.P.Cram, A.C. Brown III, E. Freitas, and R.E. Majors, Paper 115, Pittsburgh Conference on Analytical Chemistry and Spectroscopy, Cleveland, OH, March 5-9 (1979).

31. R. E. Majors, E.L. Johnson, S.P.Cram and A.C. Brown III, Paper 116, Pittsburgh Conference on Analytical Chemistry and Spectroscopy, Cleveland, OH, March 5-9 (1979)

32. R. E. Majors, The 25^th Anniversary Issue, Supplement to LCGC No.America, 25, 18-22 (2007).

Figure captions

Figure 1: Schematic of a homemade LC system.

Figure 2: First bottle of a narrow-distribution (5-10 µm) microparticulate silica EM gel, type Si60

Figure 3: H vs. v (van Deemter) curves for azo dye as a function of particle diameter

Figure 4: Ron Majors working on 8500 dual syringe pump system (circa 1975). Note the absence of safety glasses.

Figure 5: Ron Majors presenting poster prize to Isabelle Francoise at HPLC 2007 in Gent, Belgium.

Figure 6: Celebrating the shipping of the 1000th Varian LC 5000 HPLC Instrument. Left to right: Ron Majors, Paul Batchelder, Robert Graf, and Urs Christen.

Figure 7: A typical frustrated Chemist (and a younger, mustached Ron Majors) looking for a better way to do sample preparation.

Figure 8: First bottle of a narrow distribution (5-10 µm) microparticulate silica EM gel, type Si60

Table 1: See Word document uploaded to Censhare.