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An “Accidental” Career in Science L. R. Snyder “Autobiography is only to be trusted when it reveals something disgraceful. A man who gives a good account of himself is probably lying …” George Orwell Benefit of Clergy: Some Notes on Salvador Dali Have I lied? Let the reader judge. The Early Years I was born in Sacramento, CA, in 1931, and lived there until entering college in 1948. At the age of nine I had actually decided on a career in chemistry, the result of a chemistry set for Christmas from a favorite aunt. What fascinated me at the time was that stuff called compounds could be predictably assembled from things called atoms. This concept was presented in jig-saw form. An atom of oxygen had two notches in a circular representation (two missing electrons), while each hydrogen had a single projection (an available electron). So, two hydrogens plus oxygen equal water: oxygen + hydrogen + hydrogen = water From this the stoichiometric nature of chemical reactions follows. After this initial epiphany, my zeal for chemistry paused for a few years, until for the first time I had easy access to – and interest in – a public library. So I started reading chemistry books, and at that age I could comprehend the basics without too much difficulty; I was also absorbing a lot of chemical facts and history, the sort of stuff that has (regrettably) largely been removed from high school and college courses. Within a few years I had mastered the equivalent of a high-school chemistry course on my own (theory only), and was then able to assemble my own chemistry lab in the basement of our house. During the next three years, there were several lab-related events – any of which might have ended my career before it started. To recall a few:

Fe2O3, CoCl2 HNO3

O H H H O H

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• an experiment to produce nitroglycerine that went very wrong • the discovery that HCl plus KClO4 does not produce Cl2 (my first

conclusion), but the highly explosive Cl2O7 • numerous recreational “experiments” that involved the combination of

sodium and water • mistaking kerosene for water when transferring white phosphorus from

one container to another I could have burned down our house, lost my sight (came very close) , and maybe been arrested for vandalism. I doubt that my eventual career in chemistry would have benefited from any of these possible outcomes. And yet I pity the present generation whom we have very carefully “protected” from encounters such as these, both at home and at school. An experiment to produce nitroglycerine The day I decided to make nitroglycerine, I followed directions from an 1898 encyclopedia. Everything seemed to go well, until it was time to pick up the mixture of nitroglycerine and acid from an ice bath. But the container slipped as I was lifting it, followed by a sudden change in color of the mixture from clear to dark brown. This gave me about a second to turn around and run from the lab bench, whereupon the mixture exploded – spraying acid and broken glass everywhere. Several test tubes that were located opposite the lab bench were actually shattered, but fortunately I escaped any noticeable injury.

As no one was home, and it appeared that I was unharmed, I cleaned up the mess and kept quiet when my Mom and Dad returned. The next day Mom approached and asked: what happened to your shirt? Only the neck and sleeves were left! The rest had dissolved in the wash because of the acid that splashed on it. I confessed that an “accident” had happened in the lab, without going into detail.

College Years In 1948 I entered the University of California at Berkeley as a chemistry major. At the end of my sophomore year, I had a blind date with my future wife Barbara Sheppard.

?

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We became engaged a year later, and married after we each graduated in February of 1952. Barbara has proved to be the ideal helpmate, complementing me in every respect, maturing and further socializing me over time, and becoming more and more an object of my affection as the years have passed.

Don Noyce I took on a senior research project in my last undergraduate semester under the guidance of Don Noyce, who would then become my graduate advisor. My first impulse is to say that the senior project didn’t work out very well, but in fact it showed that the initial approach based on titration wasn’t very promising – hence saving some time during subsequent work on this problem. The work then continued in a new direction (with UV detection) throughout my 2.5 years of graduate school. The research topic was: why does the aldol condensation of benzaldehyde with methylethylketone yield one isomer under acidic conditions, and a different isomer under basic conditions: C6H5-CHO + CH3COC2H5 ! C6H5-CH=C(CH3)COCH3 (acid) ! C6H5-CH=CHCOC2H5 (base) My first semester as a grad student had its ups and downs, mainly downs. Kinetic studies were carried out by following the appearance of product as a function of time. The concentration C at any time of the initial reactant benzaldehyde could then be assumed equal to the initial concentration C0 minus the product concentration. However resulting plots were complicated by an unanticipated shape:

Then there was the problem of two sets of broken quartz spectrophotometer cells ($300 each), as well as my taking leave for a summer job without discussing this beforehand with Noyce (I thought grad school was like college, with summers off). On returning that fall he was understandably irritated, and suggested that I might be better off with another project and advisor. I strongly demurred, Noyce was basically an easy going guy, and he granted me a provisional stay.

x x x

log C

4 8 12 (hrs)

Induction period

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Our initial results eventually suggested that an intermediate was involved (an alcohol, prior to loss of water to yield the final product). Soon after I was able to isolate the intermediate, and my research project and relationship with Noyce then improved considerably. While we never really answered the question of why different isomers are formed at different pH, we did come up with a separate, fairly interesting conclusion: the application of the Hammett σ-ρ approach to 2-step reactions can appear to break down, even though it works quite nicely for the individual steps. Life in Industry Begins During my final months in grad school, I interviewed for a job at just two companies (at that time, a university position did not occur to me). My final choice – without much reflection – was a job with Shell Oil, located in a shed in the middle of their Houston refinery. So in 1954, off we went … had I gone with another company, it’s likely I would have entered a much more “structured” research environment, with less adaptability to my limitations and little opportunity to find my way on my own. Instead I became part of a small research group whose goal was the compositional analysis of petroleum. Despite the size of the group, and its location in the “backwater” of a refinery, it could be considered “cutting edge”. The group leader was M. J. (Jack) O’Neal, one of two people who separately developed high-molecular-weight mass spectrometry for organic analysis in the early 1950s. By 1954 his group led the world in the analysis of higher-boiling petroleum fractions (those above the gasoline range), despite initial resistance from the main Shell research group in Emeryville, CA. About 1950, Jack had suggested a research program aimed at the first-time development of mass spec for the analysis of high-molecular-weight compounds (and especially petroleum). At that time, the head of the Shell research center at Emeryville was a well regarded expert in mass spectrometry. Jack’s proposal came to his attention, and his comment was: “He will never be able to make this work, and if he does, it will never find any use”. Jack boot-legged the project, and the rest is history. So much for “expert” opinion. The mass spec analysis of petroleum fractions boiling above the gasoline range required the prior separation of these samples according to compound type. Shell had very crude procedures for separating petroleum samples by adsorption chromatography into three different fractions: saturated hydrocarbons, aromatic hydrocarbons, and so-called “resins” or (ill-defined) heterocompounds that contain nitrogen or oxygen. Chromatographic columns were packed with alumina or silica, and eluted with hexane or benzene. The saturates and aromatics fractions were then analyzed by mass spec.

Resins aromatics saturates

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When I arrived at Shell, they were interested in more effective separations by compound type, in order to improve the subsequent mass-spec analysis. One approach was to improve the selective complexation of aromatics by the use of π-acceptors such as trinitroflourenone, and I suggested that we synthesize more highly nitrated aromatics. This idea would prove problematic, as the reaction involved a nitration at a temperature below 20oC, with slow addition of nitric acid. The circa-1940s reaction flask I used had a ground-glass valve that happened to freeze during addition of the nitric acid. While fumbling with the valve, I noticed that the temperature of the flask had risen to about 80oC, accompanied by vigorous evolution of NO2. I quickly closed the hood, and waited for the excitement to die down. Unfortunately the outlet of the hood (at ground level) fed into the air intake for the next building, from which about 50 people quickly erupted … Jack was not too happy. My Introduction to Chromatography In 1955, Mac Simmons, Jim Ogilvie and I were told to set up a GC system in the lab, using the galleys of a text by Keulemans for direction (until then, I hardly knew what chromatography was). This was to be followed by studies of “high-temperature” GC. I played a minor role in the actual assembly of the equipment, but I still remember puffs of smoke leaving the column as successive wax components were eluted. As we collected retention data, I soon began noting a relation between retention time and solute structure, and then used this for my own derivation of the Martin equation (which I would not actually read about for another 6 years).

RM ≡ log k ≈C + ΣΔRM ΔRM refers to the contribution to retention of different solute functional groups. But above all I was struck by the power of this new technique (GC) that could so easily separate complex mixtures, as well as easily generate fundamental data for further analysis by the principles of physical-organic chemistry (my doctoral area, as well as the basis of the Martin equation). This contributed to my future interest in understanding and applying retention in chromatography. Al Zlatkis, a future impresario of chromatography, was also part of our group at that time. I recall Al speculating about what GC might do if million-plate columns could ever be packed. He was actually the first to achieve this goal (with un-packed capillary columns), in his later position at the University of Houston.

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Before I left Shell in 1956, we were asked to develop a GC analysis of gasoline samples for three 6-carbon compounds that are interconverted during refinery processing: methylcyclopentane (MCP), cyclohexane (CH) , and benzene. As gasoline is a reasonably complex mixture, and GC plate numbers in 1955 were quite modest by today’s standards, the required analysis could not be achieved with any of several different columns that we tried. But as I considered the retention-time data for a polar vs. a nonpolar column, it became clear that if a C6 fraction from a nonpolar column were diverted to a polar column, the desired separation could be achieved (see below). I didn’t realize it then, but this was an early example of column switching (and note the final resolution of the three target compounds!). gasoline leaving nonpolar column C6-fraction diverted to polar column

_____________

3 compounds of interest

Leaving Houston After two years of the Houston weather (somewhat uncomfortable by California standards), Barbara and I were prompted to return to California by an ad in C&E News. The ad described a position with the Technicolor Co. in Burbank, CA. They wanted someone to carry out studies on the kinetics of film dying, and they offered a quite nice salary with a three-year contract. Their goal was to better control the production of film by means of the dye-transfer process, so as to make the manufacture of Technicolor movies more reproducible and economical. This fit with my interest in physical-organic chemistry, so I responded to the ad and was eventually offered the position. Technicolor: My Second Job After arriving at my new job, I soon set up a simple apparatus to mimic what happens during film dying, for a better understanding and control of the manufacture of moving-picture film.

10-psi to dye transfer

C6

C6

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An ordinary 5-gal water bottle was used to hold and pressurize the dye solution for delivery to a film holder, where dying of the film took place under controlled conditions. While the approach was rudimentary, it pretty much did what was needed … except for the occasional exception, illustrated by the following example. One day while our dyeing experiments were underway, a dapper young man from the next lab was there for the summer … while waiting to go on to Princeton as a grad student. His clothing was always immaculate, and as he sauntered over to where we were working (in front of the dye tank), he asked in his heavy French accent: “Snyder, tell me how eez it you manage to stay zo clean while working wiz all of zees dye zolutions?” As I pondered an answer to his question, my glance fell to the front of his body, where a pin-hole leak from the tubing was spraying dye all over his pristine, well pressed pants. Before I could answer his question, his eyes followed mine, whereupon he quickly left the lab. Of course, during our work I and my assistant did not entirely escape an occasional encounter with dye … but we wore lab coats over rather ordinary clothing. The first year at Technicolor went reasonably well in terms of the applied research, including an observation of more fundamental interest. At that time there was a spirited controversy in the literature over the mechanism of dying of cloth (or film for movies) by ionic dyes. One school of thought argued that the process was driven by the Donnan equilibrium, another by adsorption on ion-exchange sites within the film. Our kinetic studies on film dying proved more compatible with adsorption than Donnan equilibrium, but the proprietary nature of our work precluded its publication. Business reverses in 1957 led to financial constraints, and the company was unable to honor the increase in pay specified for the second year of my 3-year contract … although I could stay on at the old salary. By then, however, I had become aware of other limitations of this small, very applied R&D group, as well as related questions about a professional future … so this allowed me to escape the contract requirements and head for what would prove to be a more promising job. Union Oil Research Center Union Oil and Adsorption Chromatography After a short job search, I was offered a position with the Union Oil Co. of California at their Brea research center. At that time, many oil companies were just setting up programs for the

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compositional analysis of petroleum – along similar lines as at Shell Oil in Houston. Union Oil took note of my experience at Shell and felt that I could accelerate their program. This could not have happened by simply transferring from Shell to Union. In those days, one oil company did not hire staff from its competitors (gentlemen’s agreement). I had to move first to Technicolor, then to Union Oil … not my original plan. Prior to making me an offer, my soon-to-be manager George Lake contacted Jack O’Neil at Shell to check out my prior experience. I heard later that Jack recommended me for the job, as long as they kept me out of the lab; George accepted the recommendation, but ignored the advice. During my further time at Union Oil, I managed to avoid both out-of-control chemical reactions and other noteworthy accidents (and after Union I seldom worked in the lab). A side note. My new job at Union Oil would be my third in just three years. This occasioned a note from my grad school prof, Don Noyce, that maybe it was time to settle down (a quite reasonable suggestion). Back to Union Oil. When I arrived at Union Oil, they had a very competent mass spec lab headed by Ev Howard, a 45-year-old high-school graduate. I had learned from my experience at Shell that the main limitation in the analysis of high-boiling petroleum samples was the need to adequately simplify the sample prior to mass spec analysis. My first proposal – after being assigned an office and lab space – was a request for a month to carry out a literature search on adsorption chromatography, in order to improve the separations that were needed prior to MS analysis. Being fairly new to the game, with few ideas of their own, my management agreed. My subsequent reading in the well-equipped Union Oil library uncovered many past attempts to develop a science of adsorption chromatography; i.e., one that could predict the effects on retention of sample molecular structure and experimental conditions – and therefore allow separation to be systematically optimized. While these pre-1957 attempts were suggestive, it became clear that retention in adsorption chromatography usually varied with sample size. At the time I did not appreciate that this was less true for TLC separation, where ambient humidity normally deactivates the adsorbent. With retention dependent on sample size, however, I saw no possibility of any systematic treatment of these column separations. So I began to examine the retention of various petroleum-related compounds with alumina as adsorbent. These first chromatography experiments involved (a) collecting

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small fractions from each experiment (with a single compound injected), (b) tabulating UV values for each fraction, and then (c) plotting the %-eluted vs. cumulative volume and assigning a retention volume based on 50% elution of the sample from the column (by interpolation on “probability” paper).

(a) (b) (c)

A few experiments demonstrated that linear isotherms and constant retention volumes (R, mL/g of adsorbent) were achievable, for sufficiently small weights of sample. This was only possible because of the sensitivity of UV detection for the aromatic hydrocarbons I was studying (I was using the same model Beckman DU spectrophotometer that enabled my work in grad school). Some applications of adsorption chromatography ` By carrying out adsorption chromatography on petroleum samples under small-sample conditions, it was also possible to prepare narrow fractions of different compound types that could be collected on a timed basis and analyzed by UV (so-called “linear elution adsorption chromatography” or LEAC).

Several samples could be separated and analyzed simultaneously. Much of the experimental work during my time at Union Oil was carried out by my technician, Forrest (“Timber”) Wood. Timber was a lot of help and a good friend until his death in 1972. Assays by LEAC for various petroleum compound classes (aromatic hydrocarbons by ring number, olefins, alkyl and aromatic sulfides, pyridines, indoles, carbazoles, etc.) were soon developed and published. This mix of basic and applied research was very productive. It also resulted in our

R

sample weight/adsorbent weight

LEAC equipment

Fraction # 13 16 19

UV

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discovery of the predominance in crude petroleum of 3,4-benzcarbazoles (vs. the 1,2- and 2,-3 isomers) – and their resemblance to the structures of certain plant alkaloids from which part of petroleum was presumably derived millions of years ago. a di-alkyl, 3,4-benzcarbazole The “Universal Method” Although we had developed procedures for separating petroleum samples prior to their mass analysis, the need for the separation of each sample increased the time required for analysis by at least an order of magnitude. So in 1966 we turned our attention to the elimination of the separation step. This became possible because we could now isolate relatively pure fractions of the different compound types present in these samples (saturated hydrocarbons, benzenes, di-, tri- and tetra-aromatics, etc.). With fractions from samples of different boiling point or molecular weight, their mass analysis generated data that could be used to establish the coefficients in a multi-factorial regression. This in turn permitted sample analysis by mass spec without prior separation, greatly expanding the number of samples that could be analyzed each day. The procedure was referred to within Union Oil as the “universal method”, because it could be used for any sample. This assay procedure was a sufficiently novel and valuable technique that Union was able to license it to another oil company. To my knowledge, this was the first time one oil company had paid another for the use of an analytical method. Theory of adsorption chromatography Once retention data had been collected for a sufficient number of structurally related aromatic hydrocarbons, it became possible to again apply the Martin equation for the prediction of retention as a function of compound structure, and then to expand the model to include the effect of the mobile phase and adsorbent on retention. Because of the tediousness of measuring individual values of R (equivalent to k), general conclusions based on a relatively small number of experiments (i.e., several hundred) could only be reached within the framework of a model grounded on physical-organic chemistry. This model assumed a competition between molecules of solute (S) and eluent (E) for a place on the adsorbent surface. x

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It was also found that solute-solvent interactions in the mobile phase are much less important than corresponding interactions in the stationary phase, and can therefore be ignored. The model could then be reduced to a simple equation that related retention to the solute and the mobile phase:

log k = S0 – As ε Here k is the retention factor of the solute, S0 refers to the adsorption energy of the solute, As is the relative area of the solute molecule, and ε is the adsorption energy (or strength) of the solvent (mobile phase). The effects of a change of adsorbent (e.g., silica vs. alumina) could be handled by the use of different values of S0 and ε for various solutes and solvents. Retention also varies with adsorbent water content, which can be accounted for by introducing an adsorbent-activity parameter α:

log k = α(S0 – As ε) The latter linear-free-energy relationship is fairly simple, and it ignores certain second-order contributions to retention. However a comparison of experimental values of k with this equation then allowed such secondary effects to be determined. Further comparisons of experimental vs. predicted differences in log k (i.e., “secondary effects”) as a function of solute structure and separation conditions then led to a more complete understanding of these contributions to retention (usually the result of steric and/or electronic interactions, as well as the “localization” of polar solute functional groups on strong adsorbent sites). During the rest of my time at Union Oil I was able to create a comprehensive and self-consistent picture of adsorption chromatography (mainly concerning retention rather than column efficiency) which culminated in my 1968 book: Principles of Adsorption Chromatography. A summary of our development of a theory of adsorption chromatography:

• initial application of the Martin equation, with satisfactory results for hydrocarbon solutes

• adsorption sites must be covered either by solute or solvent molecules, therefore retention involves a competition for a place on the adsorbent surface

• interactions of the mobile phase with either solute or solvent molecules are much less important than interactions of solute and solvent with the adsorbent; consequently mobile phase interactions can be ignored

• if we disregard entropy effects, ΔGo must equal the adsorbent interaction energy of the solute molecule Ex minus that of n eluent molecules Ee displaced by the adsorbing solute molecule, resulting in ΔGo = Ex – n Ee, or log k = S0 – As ε

• the Martin equation, which becomes S0 = ΣQi for different solute groups i, was found to consistently fail for certain solutes, suggesting some new effect not previously considered; this led to the discovery of solute localization

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• similarly, the strength ε0 of a pure solvent should equal S0(solvent)/As(solvent), except for more polar solvents that also localize; accurately predicted values of ε0 for all solvents are obtained when solvent localization is taken into account

• finally, localization of both solute and solvent molecules results in major, predictable changes in solvent selectivity for polar solutes (a critical tool for method development when using adsorption chromatography)

An aside. I often visited our on-site library, usually looking for a particular journal. On entering the library, I would ask for its location, then spend 5 to 10 minutes

fruitlessly searching, followed by asking the staff for help. I have always had a problem in finding things, probably because I start with a fairly precise (but usually wrong) mental picture of what I am looking for, and then expect an exact match. After a half-dozen or so of these encounters with library staff, from then on when I entered the library with a request for the location of some item, one of the ladies would lead me to the book or journal.

Some related projects While my main interest during this time was the theory of adsorption chromatography and its application to the analysis of petroleum samples, there were other opportunities to apply chromatography to various problems that we were asked to solve; for example: Failure of a pilot plant. In 1966 our catalyst group had been operating a commercial-scale pilot plant as part of their development of a new “hydrocracking” process for petroleum refining. After 18 months of operation, the multi-million-dollar unit suddenly became inoperable due to the buildup of a strange deposit throughout the system. Our assignment was to (a) determine what the deposit was and (b) develop assays for its presence in samples from the pilot plant. Within less than a week – as a direct result of our ongoing research into adsorption chromatography, plus the support of Ev Howard in the MS lab, we had established that the deposit was a complex mixture of highly-condensed aromatic hydrocarbons such as coronene and ovalene. Before the week was over, we were also able to develop LEAC assays for both alkylcoronenes (a precursor to the deposit) and “ovalenes” (actually a mixture of large aromatic molecules). This quickly became a very busy project for my lab. At that time if a sample was “urgent”, it was customary to attach a red tag. I still recall some samples arriving in my lab at this time with three red tags (i.e., very high priority).

coronene ovalene

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It eventually was learned that the zeolite catalyst used for the hydrocracking process had pores that were too small to allow the entry and break-down of these very large aromatic molecules within the catalyst. As a result, these compounds were continually formed and accumulated in the circulating reaction stream until they precipitated out. The continuous removal of a small side stream (with associated ovalenes) from the (re-circulated) residue eventually solved the problem. A $6,000,000 crisis. A few years later another problem came up, again with a high level of attention from upper management. Union Oil at that time had a substantial agricultural chemicals (“agchem”) business, based mainly on nitrogen fertilizers. It turned out that the company had been selling ammonium sulfate to a group of farmers in Idaho who were growing a new potato hybrid. About seven years are required before a new potato variety enters the market, in order to produce enough seed material for distribution. This particular crop was in its sixth year, so represented an investment of a lot of money. Well, after the most recent application of Union Oil’s ammonium sulfate, all of their potatoes died! A sample of the fertilizer quickly arrived in my lab, and we confirmed that it could kill plants. After an organic extraction and recovery of the residue, a quick GPC run showed an unexpected peak of intermediate molecular weight. The latter was then identified by mass analysis; the guys in our agchem department were then able to confirm that the compound was a herbicide that was NOT available for sale. To make a long story short, the only company making this herbicide (as a chemical intermediate) had supplied Union Oil with the sulfuric acid to make our fertilizer! After seeing our data, the other company’s lawyers immediately assumed responsibility: problem solved (for Union Oil). Petroleum Nitrogen and Oxygen Compounds After about 10 years at Union Oil (in 1966), most of our original goals for petroleum analysis had been realized. As I glanced over the remaining gaps in our knowledge of the composition of petroleum, it became apparent that not much was known about the nitrogen- and oxygen-containing compounds in higher-boiling petroleum fractions. As these compounds represent trace components embedded within an extremely complex mixture of hydrocarbons and sulfur compounds, it is not surprising that a detailed account of these “hetero compounds” had not yet been achieved.

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The approach I proposed for addressing this problem was based on our ability to predict the retention of different compound classes in petroleum as a function of separation conditions. This suggested a multi-step separation procedure where each of ~50 resulting fractions could be predicted to contain only certain possible compounds (ion- exchange separations not included, and each box can represent several fractions): Each fraction was then analyzed by mass spec, UV, and IR. Combined with our knowledge of which compounds could occur in a given fraction, the first qualitative and quantitative analysis became possible for all of the nitrogen- and oxygen-containing compounds present in significant amounts in one crude petroleum distillate. My associate Bruce Buell played an important role in this project, which reminds me of another example of my impatience and clumsy fingers. Bruce was an amateur rock collector, and occasionally carved some of his finds into nice trinkets. One day he proudly produced one of these for my inspection, and in my eagerness to see it I managed to drop and break it (manual dexterity and patience have eluded me throughout my life). Both of us were devastated, and the only thing I could think of to partially remedy the situation was to offer him a copy of my 1968 book, which has just been published. I am not sure that this made much in the way of amends, but Bruce accepted it in good grace. The Beginning of HPLC at Union Oil My inadvertent introduction to HPLC occurred in late 1966. I was almost finished with my first book, and I thought something should be included on peak width, in addition to the main story on retention. Band broadening was not very important for the compound-class separations I had been working on, many of which could be implemented with a few hundred plates. So in 1966 we assembled some equipment into a prototype system that allowed adsorption chromatography to be carried out in semi-automated fashion with a

&*!#%

Aliphatic Aromatic N, O cpds N, O cpds

Petroleum fraction alumina

Saturates, Alkylnapthalenes, PAH’s alkylbenzenes alkylsulfides N, O cpds

silica

Alkylnaphthalenes Alkylsulfides PAH’s N, O cpds charcoal

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commercial RI detector, pressure pumping, and a crude, home-made sample injector (a “rig” similar to the one we had been using for GPC). Because we were working for an oil company, solvent consumption was not an issue (nor was sample size). Consequently the first equipment featured wide-diameter columns … quite the opposite from what Csaba Horváth, Josef Huber and Jack Kirkland were doing at about that time. These wider columns were easier to pack, and they largely eliminated problems with extra-column band broadening. This greatly simplified the design and construction of the resulting chromatographic system. My initial goal was simply to measure plate number as a function of mobile phase velocity and the particle size of the alumina column packing. However, the first separation (a) seemed impressive for the time (N = 4200), if requiring 4 hr to complete! (a) (b) These same studies also allowed some interesting predictions (b) on what kind of plate numbers could be obtained with smaller particles and higher pressures. This work was described in Analytical Chemistry (1967). Once this work was completed, its practical application in our lab followed, as illustrated by a 1968 assay for our catalyst department of hydrogenated quinoline (HQ) samples: a modified UV-spectrophotometer was used as detector. We were also using gradient elution at this time as a tool for HPLC method development. These first experiments led to a further study of HPLC during my final four years at Union Oil. A very important concept that emerged during this time was the equivalence of isocratic and gradient separation, based on similar values of k (isocratic) and k* (gradient). This work with Dennis Saunders eventually proved important in the development of the linear-solvent- strength model of gradient elution (see Linear-solvent-strength model, p. 24). In 1969 a GC meeting in Las Vegas was broadened to include several HPLC presentations. This may have been the first meeting where most of the “movers and shakers” in HPLC first gathered together.

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I was sufficiently stimulated by this meeting to later organize a half-day HPLC session at a local college (Cal State-Fullerton), for a review of some of these presentations. It was initially estimated that 25-30 attendees might be interested, but as registrations for the meeting piled up, the original room was successively changed to a larger room, then a full auditorium. Eventually about 150 people showed up; interest in HPLC was definitely growing! This “small” meeting was successful enough to repeat each of the following two years while I was at Union Oil. In 1971 speakers were brought in from all over the US, including Jack Kirkland, Csaba Horváth, Cliff Scott, and several others now forgotten. Csaba gave an amusing talk on his experiments at 15,000 psi … as the column was pressured up, there was a sudden crunch, with a complete stoppage of flow due to collapse of the column packing. It would be another 20 years before pressures this high were again attempted. I left for my next job shortly after that meeting, but in following years the meetings were continued in the Bay Area, under the guidance of Ralph Jentoft and Hoc Gouw from Chevron (Richmond, CA). These meetings may have eventually influenced the formation of the California Separation Science Society (CASSS). My Next Move By the end of 1970, I realized that I had no more questions about petroleum composition or its analysis. I also saw that HPLC was likely to become an important technique, but there would be a very limited opportunity to apply it within the oil industry. In the spring of 1971 I was approached to become director of separations at the Technicon Corp. in Tarrytown, NY. The job description was nebulous, but there was a 50% salary increase and the promise of lots of resources … and I was ready for a change.

Morris Shamos My new boss was Morris Shamos, who had recently left his job as

head of the physics department at NYU. Morris and I enjoyed an excellent relationship until his death in 2002; he was well known nationally, to the extent of meriting an obituary in the NY Times. The next 11 years at Technicon played an important role in my career development, but not always for the usual reasons. Company financial problems arose about the time I was hired, so the planned separations department lasted only six months and

never amounted to much. Within a year I was appointed VP of research for the company, and a year later VP of clinical chemistry. Technicon’s business was automated equipment for clinical labs, based on the AutoAnalyzer (segmented-flow) technology and various

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colorimetric or enzymatic reactions followed by UV-visible detection. At that time, Technicon dominated the clinical analyzer business. So my primary responsibilities at Technicon no longer included chromatography … until 1976 when I would begin a small clinical-HPLC project on the side. Evaluating outside technology. One of my initial assignments in 1971 was to visit different groups and companies in search of new technology that Technicon might purchase (they had an excess of cash and were interested in diversification). One such visit was to the lab of Nobel laureate Gerald Edelman at Rockefeller Institute, to look over his elaborate, automated procedure for separating proteins. However it was clear that the scheme was too specialized and user-demanding to have any prospect of commercial success. I recall that the company owner and CEO (Edwin C. “Jack” Jack Whitehead Whitehead) was disappointed in my assessment. Nevertheless, Jack and I enjoyed a good relationship throughout the time I was at Technicon, and for some years after. While many of the employees seemed frightened of Jack, he respected people who could forcefully defend their positions. I was seldom lacking in forcefulness, often buttressed by ignorance. Another visit of this type was to a small company with a prototype HPLC under development. When the head of the company started to demonstrate the equipment, smoke immediately began to rise from the motor. He quickly turned the unit off, which concluded our visit. Perhaps the most interesting interview of this type involved a visit in 1972 from Norman Haber, who claimed to be the inventor of a new electrophoresis technique: electromolecular propulsion (EMP). He brought a simple, iPad-like apparatus with him and demonstrated the separation of the components of a dye solution. The dye components moved apart in a few seconds, which was impressive. However he refused to discuss any details of how the separation took place, which eventually stalled negotiations. Haber returned a few years later, but again refused to disclose any details. I was suspicious of both the device and Haber, so there was still no deal with Technicon. As of 2015, there is no indication that this technology ever made any practical contribution to molecular separation.

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Off-line HPLC (and related projects) The period 1971-6 represented an important gestation stage in my chromatography career, despite spending almost all of my working hours on clinical chemistry R&D. The ACS short course. Before leaving Union Oil in 1971, I had organized an HPLC

short course with Jack Kirkland, who proved a superb fit as co-lecturer. The course was oversold in its first offering in June 1971 (Chicago), and racked up audiences of 50-100 people (and occasionally more), 4-8 times a year for the next 22 years.

The course focused my ideas about HPLC, encouraged a continued interest in the literature of chromatography, and even led to a few papers, as well as the first edition of Introduction to Modern Liquid Chromatography (1974).

Another book. An Introduction to Separation Science, written with Barry Karger and Csaba Horváth, appeared in 1973. The idea for the book started in 1968, when Barry approached me at an ACS meeting with the proposal to be a co-author. Before agreeing I looked over some of the proposed topics (crystallization, foam separation, electro- phoresis) and told Barry that I ought to read a book on separations, not write one! He persevered, however, and eventually the three of us started writing chapters. There were frequent meetings on the book, especially after I moved to New York in 1971, but eventually more time was spent critiquing each other’s work than writing new chapters. We finally realized that we all had similar (relatively narrow) interests, so we decided to add additional collaborators to the book. My memories of these meetings now feature the humorous back-and-forth; I remember that at times my sides really (I mean really) ached from laughing. Csaba could be a very funny guy, and Barry was a good straight man. The solvent-selectivity triangle. One of the more interesting of my few papers from this period was a 1974 J. Chromatography article on the solvent-selectivity triangle. x x

Jack Me

Acceptor

Polar Donor

19

The above diagram was given a prominent place in the first edition of Introduction to Modern Liquid Chromatography. This arrangements of different solvents according to properties that affect selectivity allowed solvents of similar selectivity to be grouped (circles in the above figure); solvents from different groups could then be selected as a means of changing selectivity. This later became a practical tool for use in method development. A few years later the triangle stimulated Jack Kirkland and Joe Glajch to develop their famous 4-solvent optimization procedure for reversed-phase chromatography; this soon evolved into the first commercial equipment for “automatic” method development (Du Pont’s Sentinel system). There is an interesting story behind the triangle paper. In the early 1970s, Rohrschneider in Germany was publishing on GC solvent characterization, and he had reported an extensive table of measurements of values of K for the vapor-liquid distribution of 6 test compounds using 80 different solvents. He gave a talk on this work at a meeting in 1972 which was also attended by another scientist whom I will refer to as “X”. At the end of the session, “X” mumbled to me that Rohrschneider’s talk didn’t seem to make any sense. This might have been reason enough to explore further and eventually arrive at the solvent triangle, but in addition “X” and I had some prior history, and I experienced him as stubborn and somewhat arrogant ( “X” may well have had similar feelings about me). Anyway, all of the subsequent calculations for the triangle paper were eventually carried out on a slide rule during evenings at home. One night while working on this paper, my wife Barbara remarked: “Do you really need a second job?” (work on books and a few papers at night and weekends, plus the short course) . . . I did if I wanted to stay active in the field of chromatography. Another triangle episode took place about 5 years after the paper was published. At that time Hans Poppe wrote me that his group was having trouble reproducing the numbers in my triangle paper. After checking over my original calculations, I found that a small correction was noted in the original paper, but was not included in my published calculations. This was embarrassing, and all I could do was send in another paper that corrected the original paper. The resulting triangle did not look too different, and for all practical purposes either version of the triangle was adequate. But carelessness was a not infrequent problem throughout my career … too fast, too fast! Fortunately most errors during model building are easily recognized (unless the errors are minor, as in the above example). The latter diagram was featured in the second edition of Introduction to Modern Liquid Chromatography (1979). x x x

Basic

Acidic Dipolar

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This was not the end of the evolution of the triangle, however. Ten years later, Pete Carr’s group noted that the Rohrschneider test solutes were not ideally suited to separate the three interactions on which the triangle is based. By substituting solvatochromic parameters (α, β, π*), a final version of the triangle resulted in which the placement of solvents more accurately reflects their different interactions. This final version of the triangle also appeared in the third edition of Introduction to Modern Liquid Chromatography (2010). Understanding dispersion in segmented flow. While my experimental work in chromatography from 1971-1976 was pretty minimal, I did become interested in the sample dispersion that takes place during segmented flow – the technology on which Technicon’s business was based. During my first year, before taking on the job of research director, I designed a number of experiments where dye was injected into an air-segmented stream of water, followed by measurement of the spreading of the dye from one segment to another as a function of various experimental conditions. I was very fortunate to have the help of Harvey Adler (a long-time Technicon employee) in carrying out these experiments. Because a little bit of each sample segment clings to the tube wall, the sample is retarded as it moves along and eventually becomes distributed over several adjacent segments (peak broadening or sample dispersion). The extent of this peak spreading limits the rate of sample analysis, just as in chromatography, and was therefore of considerable relevance to Technicon’s business. We collected a nice set of data, consisting of measurements of both band retardation and band spreading as a function of various experimental conditions. The retardation (or retention) values are determined by the thickness of the film df (see above illustration), which in turn can be estimated in the same way as film thickness in GC capillaries: df = 0.5 π dt

(uη/γ)2/3, where dt is tube diameter, and other variables refer to the segmented stream: u is its linear velocity, η is viscosity, and γ is surface tension. But a description of band spreading was less obvious. The dispersion process is the result of so-called bolus flow through the tube, with a circular (“tank-like”) rotation of liquid that occurs within two discrete regions of the moving segment.

ACIDIC DIPOLAR DIPOLAR

21

As a result, there is not much mixing of adjacent liquid elements within each liquid segment, other than by diffusion. At first our results on band spreading resisted interpretation, but a few years later I realized that Giddings had derived an analogous equation for band spreading in (at that time hypothetical) plug flow – where the solvent velocity is constant at different points within the column. By considering the two parts of bolus flow as each equivalent to plug flow, a comprehensive equation for band spreading in segmented flow resulted. This work was later published in Analytical Chemistry, and then adapted to a lecture on “effluent storage” for the 1976 HPLC meeting in Philadelphia. I recall a few remarks from that audience, questioning whether my talk had much to do with chromatography! Giddings was also present at this meeting, and I was looking forward to his comments on our work. Unfortunately he missed that session, and I never heard his opinion. However, I often think of this work as one of my more rewarding efforts. Segmented-flow chromatography. A few years later, another application of our theory of dispersion in segmented-flow arose: the conceptualization of segmented-flow liquid chromatography (SF-LC), using coated capillaries. The idea occurred in early 1975, and soon after we took advantage of an NSF faculty grant to bring in two college professors for the summer to work on SF-LC: Bob Grob of Villanova and Prof. “Y” from the University of Vermont. Bob and I had met earlier, while “Y” had published an intriguing paper in 1970 on the use of sub-micron particles in centrifugal LC. Unfortunately, “Y” was unable to work effectively with Bob, and nothing resulted from the two months they spent on the project. In later years Bob often commented on his trying time with “Y”! “Y” Grob John Dolan and I later successfully applied SF-LC for the preparation of serum samples prior to their analysis by automated HPLC (see HPLC research, pp. 24-7). X

analyte

&*!#%

22

The idea was to separate the drug from the rest of the serum (protein plus cells) in a first SF-SEC column, with diversion of the drug fraction to a conventional reversed-phase column. Because the drug fraction ended up in aqueous solution, there was no need to remove organic solvent – as would be the case for extraction of the drug into organic (the usual sample prep approach at that time, for use with reversed-phase separation). The procedure worked quite nicely for anticonvulsant drugs, but unfortunately it was later found that the technique could not be extended to trace assays (e.g., tricyclic antidepressants). Lower-molecular-weight serum interferences that were not separated from the drug analytes during the SF-SEC step severely limited the HPLC assay of these ppb analytes. And trace assays in clinical labs were important! For this reason, the concept of segmented-flow chromatography soon became an historical relic. Two chromatography mini-projects passed through my hands during 1972 and 1973. The first task was the development of a GC system combined with automated sample extraction, for the purpose of detecting illicit drugs in serum. An outside firm (Bendix) worked with us to supply the GC system, while Don Burns in my group focused on sample prep and making the whole system work. The final product performed sort-of OK, but soon after we dropped the project. The second project was in response to an NIH request for proposals to analyze three polyamines in serum. At the time, these compounds were believed to be indicators of certain cancers. We received funding for our proposal, based on an automated extraction of the sample followed by ion- exchange separation. The project worked out well under the direction of Marve Margoshes, with Harvey Adler helping. Soon after, however, it was found that polyamines were in fact not reliable predictors of cancer, and the project was dropped. My role was to oversee each project, as well as provide technical advice. Bound enzymes. In the mid-1970s I began another collaboration with Csaba, one based on chromatographic principles – but not chromatography. At that time Technicon had several assays that required the use of expensive enzyme reagents (consumables). Csaba came up with the idea of using enzymes bound to the inside of tubing as a cheaper, reusable replacement for these reagents (with segmented flow through the tubing). The concept was successfully incorporated into Technicon’s clinical analyzers and created quite a stir when introduced to the market in 1976.

with Csaba at a meeting in 1978

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An aside. Allow me to reflect on a different aspect of the work that chromatographers (and other scientists) do. When I was a young, would-be scientist, I had this picture of how science works. We carry out experiments – either in the lab or on paper – and we publish our results. The process had been compared to the construction of a long and ever-growing wall. Each of our results has become a stone in this permanent wall of science … the work has been immortalized! The actual situation is more like the construction of real walls over long periods of time. The wall begins to be built, but in time the wall tends to settle and be covered by surrounding soil, individual stones fall out and are replaced, and further stones are added to the top of the wall. Thus most of what we do today will be like the lost or covered-up stones of the wall … for all practical purposes, gone from sight forever. Which is not to say the work was not worthwhile; at the time it helped support the wall for the addition of later stones, and it guided further work on the wall. And very occasionally one of the old, lost stones may be retrieved and reused. The Technicon laboratory After a year as research director, my department was split in two, and I chose to head the larger of the two parts. Thus I became director of the clinical chemistry department with a lab of about 30 people; during the next six years the size of the lab would almost double. During my tenure as director, the primary responsibility of my department was to develop procedures for a new 20-channel chemical analyzer: the so-called SMAC system (Sequential Multiple Analysis – Computer). The latter, computer-controlled apparatus could simultaneously analyze twenty common constituents in serum samples, at a rate of 150 samples per hour (with minimal operator intervention). Examples of these analytes include glucose, urea, creatinine, electrolytes such as Na+ and K+, various enzymes, etc. During the development of SMAC, resulting assays needed to be created, validated, problems identified and resolved, calibration samples prepared, etc. There was little need for HPLC, however. My return to chromatography would require a new project. HPLC research (1976-81) In 1976 I was able to return to part-time studies of HPLC for the development of an HPLC system to assay therapeutic drugs in serum. This then became full-time in 1978 when Tom Adams from Hybritech replaced me as director of clinical chemistry. I had been trying to recruit my replacement during the preceding 18 months (after 7 years of

24

management, I wanted to return to the lab). Tom and I enjoyed a very good relationship at the time, and a decade later I would do some consulting for his new company in Southern California, Genta (see p. 35) . At this time we were lucky to hire a small team of guys with great future potential: John Dolan (first of all), Steve Bannister, and Sjoerd van der Wal. A related HPLC program started at this time in another department at Technicon, with Russel Gant as the resident chromatographer. Russ worked closely with our group on the more fundamental studies we carried out as part of our development of clinical HPLC between 1977-82. Another important contributor to our work was Roy Eksteen, then one of Barry’s grad students who was on our payroll via a grant from Technicon. His work on perfecting the column packing process was critical to the eventual development of our drug analyzer. The linear-solvent-strength model. One important outcome of this collaboration began with my reading one of many articles where plate numbers in gradient elution were (wrongly) calculated in the same way as for isocratic separation. In response, I put together a quick note for publication, and showed it around the group. John and Russ thought that the idea should be expanded before publication, with the addition of some experimental data. To make a long story short, this soon led to the development of the linear-solvent-strength model of gradient elution (perhaps the most widely used work with which I have been associated). As a side note, the final paper made no mention of my original thoughts on plate number calculations in gradient elution! The linear-solvent-strength (LSS) theory of gradient elution did not emerge as an “overnight” concept, despite the fact that the above work at Technicon was carried out over a Christmas vacation. Rather the model represented the conclusion of 15 years of observation and reflection:

• LSS gradients have certain desirable properties (1964) • gradient steepness is equivalent to a change in isocratic values of k so far as their

effects on resolution (1969) • band width is predictably altered by peak compression in gradient elution (1969) • various aspects of LSS separation can be treated quantitatively (1980); in

particular, a gradient retention factor k* (equivalent to k in isocratic elution) can be calculated from gradient conditions: k* = tGF/(1.15 VmΔφS); thus an increase in gradient time tG is equivalent to a decrease in isocratic %B.

This effectively allowed the use of everything known about isocratic separation to be applied to gradient elution as well (in 1980, isocratic elution was much better understood than gradient elution). Over the following 30 years, the linear-solvent-strength model would lead to a comprehensive and practical understanding of gradient elution, for application to HPLC method development, the separation of large biomolecules (p. 28), and prep-LC (p. 32). All of this was summarized in High-performance Gradient Elution (Wiley, 2007), by John and myself.

25

Boxcar chromatography. Our group was highly enthusiastic and motivated, and a big moment for us was the conception of boxcar HPLC. Our HPLC assays usually involved separating one or two drugs from a complex serum matrix, which often required a large plate number (i.e., a long column) and therefore long run times. This was fatal for the high-speed assays that were needed in a clinical lab. But then we realized that if a fast initial separation could be used to partially separate the drug(s) of interest, followed by their diversion to a longer column for complete separation, several sample-fractions could be simultaneously resident within the second column, followed by their separation to baseline (another column-switching application; cf. p. 6). While 15-20 min might be required for the first sample to leave the second column, a sample could be injected every 1 to 2 min. In the example below, primidone, phenobarbital, and carbamazepine were assayed together at a rate of a sample every 90 seconds. (a) (b) (first column) (second column)

Barry Karger In (a) the fast separation on the first column is shown (with incomplete resolution of the three analytes – marked by red arrows). The latter fraction is then sent to the second column. In (b) replicate samples are shown leaving the second column (starting at about 20 min). So the overall sample throughput became quite reasonable. This work was further developed in Barry Karger’s lab with Al Nazareth. Boxcar chromatography might have become more widely applied at the time (1980), but Technicon chose to patent the technique. By the time the patent ran out, other (simpler) means for achieving high-throughput separations existed. However the technique might still prove useful for the right application (there is a description of boxcar chromatography in the 3rd edition of Intro to Modern LC).

26

Which reminds me that Barry and his group were an important part of our team from 1976-82. We contracted some of the work on our drug analyzer to Barry’s institute, and enjoyed a close relationship. As noted above, my professional relationsship with Barry and Csaba began in 1968; it remained active throughout the mid-1980s.

Barry Karger Prelude to DryLab. Another basic project we undertook, following our gradient studies, was prediction of retention as a function of both solvent strength (%B) and temperature. As might be anticipated, the relationships that described these retention dependencies were easily combined into a simple program that ran on a Texas instruments programmable calculator, so there was no question you could do it. But at that time there seemed to be little value in such predictions. Everyone then “knew” that selectivity (the primary consideration in method development) was not much affected by changes in either %B or temperature … later, during the development of DryLab (p. 30), we would learn otherwise. Setting Out on my Own So we were having a lot of fun at Technicon in the period 1976-81, and doing good work (I think). But clinical HPLC was never going to have a future for the routine analysis of commonly used therapeutic drugs in serum. More specific and sensitive assays of drugs in blood could be carried out by immunoassay procedures; machines from both Syva and Abbot became available at this time that achieved these results conveniently, relatively cheaply, and fast. In the fall of 1981, while I was on vacation in Europe with family, I got a call from Sjoerd in New York that our clinical HPLC project had been cancelled. John Dolan had seen what was coming a year before and had jumped ship to a position with IBM, who were just getting into the HPLC market. The remaining members of my team were able to connect with jobs that could use their talents, and I realized that if I stayed with Technicon it would be the end of my HPLC career. So I began to think about forming my own HPLC-oriented company – Lloyd R. Snyder, inc. – and then left Technicon in the spring of 1982. The book (1st and 2nd editions of Intro to Modern LC), lots of short courses with Jack, and numerous professional contacts became the basis of my first company. I was on the phone just before leaving Technicon, talking up the possibility of inhouse HPLC short courses.

27

My initial business plan assumed that I would be lecturing and consulting. The response to my initial solicitation for inhouse short courses was quite positive, and I quickly had enough business lined up to keep me busy about 1/3 of the first year. A consultancy at Du Pont About this time, Jack Kirkland suggested to John Schmidt (the HPLC-equipment manager at DuPont) that I might be available for work as a consultant. This led to an offer in April 1983 that was the beginning of a 7-year association with Du Pont, one that allowed me to pursue several fairly interesting research projects … this turned out to be a huge opportunity! My first assignment was to help with an immediate problem. A DuPont HPLC group (John Larmann, Joe DeStefano, Al Goldberg) had committed to a paper for the 1983 HPLC meeting at Cherry Hill: Isocratic separations of large molecules, using polystyrenes as surrogates for proteins. But they couldn’t make sense of their data. For molecules larger than about 4 KDa, small changes in solvent strength (%-ACN) resulted either in no elution of the sample, or elution at t0. The reason for this “problem” was the extreme change in k for large molecules, for very small changes in %B (Terabe had shown this in 1981 for the separation of peptides and

small proteins). I quickly organized some gradient elution experiments that pretty much confirmed the nature of the problem, and also resulted in an insightful paper … just in time for the meeting. An important aspect of this work was the demonstration that solute molecules too large to enter some of the particle pores (A and B) under size-exclusion conditions could still be retained in reversed-phase chroma- tography. This was our so-called “softball model” for the

retention of large molecules), as opposed to the “hardball model” which assumes that large molecules are not retained in small pores. Marilyn Stadalius Over the first 5 years of my time with Du Pont, almost all of the

lab work was carried out by four graduate students whom I shared with U. Delaware (Marilyn Stadalius, a student of Harvey Gold), U. Villanova (Mary Ann Quarry and Julie Eble, both Bob Grob students), and U. Pennsylvania (Barbara Ghrist, a student of Barry Cooperman). My first interaction was with Marilyn, who carried out the experimental studies for the above Cherry Hill paper. However, this initial work had ramifications far beyond

Mary Ann Quarry the isocratic elution of large molecules; it quickly led to a series of studies which for the first time rationalized the (more common) gradient separation of peptides, proteins and other large molecules (see Gradient separation of large biomolecules,

p.29).

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Selectivity as a function of gradient conditions. The work with Marilyn also led to the prediction of retention as a function of ALL gradient conditions on the basis of just two initial experimental runs. Joe Glajch picked up on this work a little later, and in some joint studies demonstrated the first example (for peptides) of marked changes in selectivity as a function of flow rate (equivalent to changes in gradient steepness, or change in isocratic %B). 0.5 mL/min 1.5 mL/min. During the first few years at Du Pont, Mary Ann Quarry and I also collaborated on a fundamental study of how equipment and experimental conditions can lead to errors in predictions of retention for gradient elution. Both Mary Ann and Marilyn continued to work on our gradient-related projects after they each had completed their PhD’s in 1984 and joined Du Pont as employees. They were able to find spare time during the day, or after hours, to carry out the experiments at work, which shows the tremendous enthusiasm and commitment they both brought to our studies. Du Pont’s tolerance for extra-curricular research at that time was also much in evidence. Gradient separation of large biomolecules. In the early 1980s, several laboratories reported unexpected results when column length was varied for the gradient separation of protein samples. In these experiments it was observed that when column length was increased, there was no improvement in the separation. Virtually the same separation was obtained, regardless of column length. In the case of isocratic elution, a longer column means a larger plate number N and an increase in resolution … as well as an increase in retention time. As seen in the protein separation on the right (mixture of ribonuclease A, cytochrome c, ovalbumin) where only column length is changed (by as much as 28-fold), there is no change in resolution and little change in retention or run time. The reason is that when column length is increased in gradient elution, a decrease in values of k* results. Resolution depends on [k*/(k*+1)] N, so an increase in column length leads to compensating changes in k* and N. These results could be rationalized by our linear-solvent-strength model of gradient elution. About this same time, a theory of “critical” retention behavior was offered as an alternative explanation of the gradient separation of large molecules. Had this been true, there would have been little possibility of improving the separation of proteins, nucleic

45-mm 6.3-mm 1.6-mm

29

acids, etc. by an increase in N – which is fortunately not the case. This resulted in an important 1986 refutation which was also somewhat polemical. The start of LC Resources and DryLab John Dolan In 1984 I was at an IBM-sponsored meeting in Arizona, where I

ran into John Dolan again. We got to talking, and he expressed some dissatisfaction with his situation at IBM; in fact, IBM shut down their entire HPLC business a little later. In any case, we decided to join together in a new company which we called LC Resources, Inc. Since then John and I have been “joined at the hip” in all of our R&D activities, and I cannot imagine where the second half of my career would have gone without him. We

complement each other in almost every way, we are both very focused, hard workers, and we are each highly enthusiastic about chromatography. We also see life in similar ways, share similar values, and have enjoyed the closest of friendships over the years. Our initial LC Resources products were two videos, one on basic HPLC and one on troubleshooting. HPLC troubleshooting was something that John was always very good at; in 1983 he had accepted the job of writing a monthly column on troubleshooting for a new magazine (LC, later LCGC). John’s very successful column has continued until the present time. Producing videos took off running, but when our first product came out in 1985 it became apparent that videos were going to be a more limited market than we had hoped. At this time, Barbara and I also returned to California (via a vacation in England). DryLab. I had been writing simple software for predicting resolution as a function of conditions – a return to the above work with John and Russ Gant in 1980 (p. 26) on predicting separation for changes in temperature and %B. John had followed these simple exercises, and suggested: “Why don’t we commercialize this stuff?” John referred to what the software did as “Drylab-ing”, from which the name “DryLab” immediately followed. In 1985 the only software we had was for predicting the effect of the column and flow rate on separation. But John quickly cobbled together workable software which we exhibited at the EAS meeting in November of that year. There was some interest at EAS, so we expanded the software for predictions that included separation as a function of solvent strength (%B) … but we still did not see this as a very promising product (in its original form and scope, it clearly wasn’t). But then came a breakthrough. After we decided to expand the software for predictions of isocratic separation as a function of changes in %B, we thought that a systematic experimental verification of the software predictions would be useful in selling the product. We had no lab of our own, so we contacted Pete Carr for help. At the time, Marty Rigney was a grad student in his group who was willing to spend a week grinding out data. We selected a mixture of alkyl and nitroalkyl benzenes as sample, and he carried out separations for different values of %B. We were greatly surprised to see that these very simple, relatively nonpolar compounds exhibited several peak reversals as %B was varied.

30

. x Two peaks overlap for 50% MeOH (indicated by an arrow), but then separate at 55% MeOH, and two other peaks (opposed arrows) reverse position between 30 and 50% MeOH. The resolution map shows still further peak reversals as %B is changed. This really opened up a new possibility: selectivity could be usefully varied by just changing solvent strength (%B) … experimentally very convenient (especially with gradient equipment). So with just two experimental runs, our software allowed the creation of resolution maps that pinpointed conditions for maximum resolution, then allowed further predictions of the effect of a change in column length, particle size, or flow rate while holding selectivity constant. These findings meshed very nicely with Joe Glajch’s results above for the gradient separation of peptides. The general theory for all of this had been formulated in my 1980 chapter for Csaba’s series of reviews (High-performance Liquid Chromatography. Advances and Perspectives), but it was not then apparent that changes in selectivity might be fairly common when gradient steepness is varied. And with further increases in column efficiency over time, even small changes in selectivity would be increasingly important. With the addition of solvent-strength variation and gradient elution, plus simulated chromatograms and resolution maps, our revised DryLab software became a much more useful product. An early gradient separation for a 15-component herbicide sample is representative of the power of DryLab (achieved with only two experimental runs). Resolution map 80-min gradient (optimum) predicted actual We were also able to incorporate much of the work I was supervising at DuPont into the further study of computer simulation and the expansion of the capabilities of DryLab. Barbara Ghrist, one of my grad students at DuPont, was quite active in this area. I still remember a poster she prepared on our work with DryLab for the separation of the 30S

gradient time (min)

Rs

50% MeOH 55% MeOH Resolution map

40 50 60 70 % MeOH

31

ribosomal proteins (18 proteins) by varying gradient shape. This represented an extremely challenging separation; however, with a 3-segment gradient (for peaks within groups A, B or C; see below) designed with the help of DryLab, she managed to resolve the entire sample (Rs > 0.8) in a run-time of 4 hrs (first time ever for this sample).

In the mid-1980s Imre Molnar invited me to Germany to give an HPLC short course with him. This continued for a few years, and during that time I introduced DryLab in one of my lectures. He quietly approached me afterward and asked: “Does this stuff really work”? That led Imre to take on DryLab distribution in Europe, which eventually became his major activity. Imre and I have enjoyed a close relationship over the years.

DryLab team at the LC Resources booth (PittCon 1992) John Lloyd Margaret Tom Imre

Margaret Watkins was our sales rep at that time. So how did DryLab start?

• development of the LSS theory of gradient elution (1980) • both isocratic and gradient separations can be simulated, if values of k0 and S are

known for each solute (1980)

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• k0 and S can be determined from two experimental gradient runs (1983) • more accurate predictions of N as a function of conditions (1983) • discovery that selectivity can be changed significantly by varying either %B or

gradient steepness (1985) Prep LC. I had given some thought to an understanding of prep LC in 1963, when we first carried out simulations on an IBM 650 using the Craig model. Unfortunately our computer at that time was quite slow, and was limited to only about 12 experimental plates within the time allowed on our company computer. However the simulations agreed reasonably well with experiments we carried out at this time. This work was sufficiently encouraging to reconsider this line of research 20 years later. Because of DuPont’s growing interest in the production-LC Julie Eble market, in 1984-5 I initiated a research program to investi- gate prep-LC with the help of one of my four grad students, Julie Eble. Like Marilyn and Mary Ann, Julie joined Du Pont after her PhD research was complete , where she worked for the next 15 years. Geoff Cox at Du Pont was also part of this project.

Prior to 1985, a practical, theory-based understanding of prep-LC was (in my opinion) non-existent, so it seemed a good time to try our luck at a reliable model. After collecting a lot of data – both computer simulations and “real” experiments – we found that our results could all be organized on the basis of some simple concepts proposed earlier by Hans Poppe and later expanded by John Knox. The effect of sample size on peak width could be described by the ratio (N/N0), where N is the plate number of an overloaded peak, and N0 is the plate number when a small sample is used. Values of N/N0 could then be related to a function (wxn) of k for a small sample (k0), the sample size wx and the column capacity ws. A single curve then resulted when values of N/N0 were plotted vs wxn for widely varying values of N0, k0, and wx/ws (an epiphany). This verified our ability to predict values of both N and peak width as a function of sample size and experimental conditions.

For moderate sample weights, and so-called touching-band (T-B) separations, it was now possible to define conditions for an optimum T-B separation. This corresponded exactly to what was needed for small-scale separations in the lab; i.e., a simple recipe for the convenient recovery of 95%+ of the desired product in 98%+ or higher purity, while

achieving maximum sample throughput.

touching-band separation

wxn

N/No

33

A corollary of this work was its extension to gradient elution. The linear-solvent-strength theory developed by us earlier showed that exactly the same relationships apply to both isocratic and gradient prep-LC, when k* for the small-sample gradient separation is approximately equal to values of k for the corresponding isocratic separation. A demonstration of this is shown next for corresponding isocratic and gradient separations (i.e., where k ≈ k*). The solid curves represent the separate injection of each compound, while dashed curves are for injection of the mixture of compounds A and B. Our work on prep-LC continued in the DuPont lab until 1989, at which point my 7-year relationship with the company came to an end. HPLC at Du Pont similarly ended a few years later, which provided Jack and Joe DeStefano the opportunity to start their own, highly successful column business. Much of our work in prep-LC used computer modeling (Craig distributions), where Langmuir isotherms were approximated by polynomial expressions. Unfortunately in one such exercise, a faulty polynomial was used that generated slightly distorted results. While the published conclusions of that study were not much affected by this (questionable) choice of isotherm, the problem was brought to our attention by a letter to the editor. Needless to say, we were justifiably embarrassed by this incident. Colleagues Some additional acknowledgements of colleagues are very much in order, for their stimulating interactions, assistance in reviewing work prior to publication, or more substantial collaborations. Our work on gradient elution benefited from the help of Peter Schoenmakers, Hans Poppe and Pavel Jandera, each of whom advanced our thinking from their own unique perspectives. Peptide and protein separations received a lot of attention during the 1980s, when Fred Regnier was a focus of much that was going on … both in theory and practice. The conversations that Fred and I had helped define for me what was more likely, and what was not; what was more important, and what was not. Prep LC also came of age at this time, with John Knox initiating our thinking in a profitable direction, and David McCalley providing stimulating ideas for the next two decades. John Knox also provided inspiration for our work on column efficiency that in turn supported DryLab. My continued involvement with adsorption chromatography over the years received a “spurt” from collaborations with Joe Glajch in the early 1980s, and was critically supported at an important juncture by Hans Poppe. Method development was very actively debated during this same period, with the work of Joe Glajch and Jack Kirkland, and Peter Schoenmakers and his group, providing an ongoing incentive to do

2.5 mg A 2.5 mg A 2.5 mg A 2.5 mg B 10 mg B 25 mg B

Gradient elution

2.5 mg A 2.5 mg 2.5 mg A 2.5 mg B 10 mg 25 mg B

Isocratic elution A, A’ B,

B’ A A’ B,

B’ A A’

B, B’

A, A’

B, B’

A A’

B, B’

A A’

B, B’

34

better. Especially pleasant from 1970 on was our close association with the entire “Dutch school”, which began with Josef Huber. LC Resources Grows In 1987 our new company experienced a 100% growth in staff (to a Tom Jupille total of 4). We were extremely fortunate to have Tom Jupille join the company as a third partner, followed soon after by his office assistant. Tom brought business skills that John and I lacked almost entirely, as well as the insights and inputs of an experienced chromatographer. The three of us soon became fast friends and very compatible business associates; this provided a basis for the expansion of LC Resources to a staff of about 30 in later years. I often reflect on how fortunate Tom’s acceptance was for the three of us and the future of our company. I also recall that at the beginning we set out some goals for our business: “have fun, contribute something useful to the world, and become filthy rich” (in that order). Depending on your definition of “filthy rich”, in the end I think we came close to each of our goals. We Start our Own Lab. The software business (DryLab) was growing rapidly, and although we had experimental support at that time from Barbara Ghrist as part of my work for DuPont, plus occasional “borrowed” time from Marilyn Stadalius and Mary Ann Quarry, we really needed our own lab if we were serious about this new business. So in 1988 we hired Dana Lommen and rented half of a lab from the local college in McMinnville, OR, where John Dolan now lived and worked.

Dana Lommen in our lab We also began soliciting customers for lab services, mainly HPLC method development, and we had spare time to carry out our own experimental research program, focused mainly on various applications of DryLab. In the late 1900s, we would move into our own, much larger quarters for the laboratory business. Consulting at LC Resources We undertook a number of HPLC related consultations during the time our company was in business. Two examples may serve to illustrate the diversity of our work in this area.

35

A legal case involving terrorism. In 1986 I was approached by phone to inquire whether I was willing to consult for a nearby law firm. The attorney at the other end of our call explained that his firm was representing a terrorist suspect in a case brought by the FBI. I quickly explained that this was not our kind of business! But the attorney persisted, explaining that while the suspect was a terrorist at the time the crime was committed, his government had subsequently changed, and that (foreign) government now wanted the charges dropped. (the “terrorist” had morphed into a “revolutionary hero”). So on that basis I agreed to participate. I should also mention that “my” law firm was paid by his country, but the US government now also had an interest in seeing that the trial should lead to acquittal (the other country has been a close ally of the US for many years). The key evidence against the suspect consisted of a tool from the suspect’s garage, that upon analysis was claimed by the government to be contaminated with an explosive linked to the terrorist act. The evidence consisted of an HPLC run which yielded a single peak with the same retention time as the explosive compound. I was asked at trial if this was sufficient evidence to identify the compound unambiguously, and of course I said “NO”. But then the courtroom was cleared of everyone except the judge, myself, and the attorneys for the FBI and my client. At this point some very recent evidence was submitted by the FBI, consisting of the original HPLC data plus a confirming mass spec. With this in front of me, I was again asked what my opinion was ... and of course I said, “that’s the explosive compound”. Now our side (the defense) objected to this last minute submittal of evidence, and after a half day of consideration by the judge, the new evidence was disallowed. The final verdict of the trial was acquittal of our client (to everyone’s satisfaction). One interesting side show took place, during the time I waited each day outside the courtroom with other witnesses (all waiting to testify when called). At that time I was carrying around my stuff in an old plastic “briefcase” handed out at one of the PittCon conferences. Suddenly one of the witnesses for the prosecution (an FBI analyst) caught side of my “briefcase’, came over to me, and asked if I was Lloyd Snyder. When I said “YES”, he asked me to autograph his copy of the second edition of Intro to Modern LC! Work at Genta. In the late 1980s, Tom Adams had called me to consult on their purification of modified oligonucleotides; these compounds were produced by Genta as possible anti-cancer drugs. Each oligo contained 10-15 nucleotides (varying amounts of A, G, C, and T), and they were currently trying to purify individual compounds by reversed-phase chromatography (RPC).

Resulting isocratic chromatograms showed that the product was strongly overlapped by failure sequences corresponding to an absence of one or more of the primary nucleotides. So LCR was asked to compare a number of different RPC columns to see if the separation could be improved. As the compounds are neutral, and acetonitrile-water was the preferred option, not much could be varied

Impure product

product area = 65%

36

apart from the column and temperature. For convenience, we were using gradients of increasing acetonitrile. So a number of columns were tried, all of which gave similar (poor) results with the usual acetonitrile/water gradients. Then for some reason I asked our lab to repeat the separation on a diol column with a reverse gradient from water to acetonitrile. This resulted in a spectacularly better separation. This is of course what most people would try today, as it was a form of HILIC (preferred for separating very polar samples, as Genta’s samples were). Further experiments based on this favorable result showed that even better separations could be obtained on bare silica (again a natural choice today). But then the project became more interesting. Isocratic separation was desired, so it was necessary to determine (by trial & error) the combination of %-acetonitrile and temperature to be used for each sample (for preferred retention; i.e., 3 >k > 10). This led us to develop a model of these separations Oligo retention times that allowed a computer to determine the best conditions for a given sample. This in turn required predicting the product retention time as a function of sample composition, temperature, and %-acetonitrile. As seen for data for several samples and temperatures, the model proved quite accurate. Only the number of each nucleotide in the desired product was needed as computer input. This work became the basis of my last patent (with several Genta employees as co-inventors). Expanding our research with help from NIH A few years earlier, I was approached by Miriam Behar, who was in charge of a study section for the General Medical Sciences institute of NIH. She wanted me to join her section, which was responsible for evaluating grant proposals from small businesses (Small Business Innovative Research, or SBIR). Serving on the study section would have involved 3 meetings a year in DC and a lot of work; as I was already quite busy, I respectfully declined. But she persisted, especially with the argument that my company could also apply for these grants, with the possibility of funding at the $500,000 level for a 2-year period (later increased to $750,000). This last observation caught my attention, and my subsequent connection with Miriam and the study section then lasted from 1988 to 2003. During this period NIH invested over $2 million in our lab – money which was largely directed to studies that I believe have had a significant impact on the practice of HPLC

Pure product

product area = >99%

37

method development. Without this support, a large fraction of the good stuff that our lab turned out during this time would never have happened, nor would the necessary continuing support of DryLab have been possible. Incidentally, having our lab in place when we first applied for NIH support proved critical to our winning that first grant … which led to the second grant, and the third, and so on. I might also say that I have known few people more dedicated to science and her work at NIH than Miriam. Everyone loved that lady. Her death in 2012, after several years of struggling with cancer, touched me and others deeply. DryLab-GC. Our first work with NIH support (1990) was the adaptation of DryLab (for HPLC) to allow GC simulations. A brief literature review suggested that a change in either temperature or temperature-programming rate should result in similar changes in band spacing (and similar opportunities for maximizing resolution) as in HPLC when %B or gradient time is varied. Our first grant was for $50,000 over a period of six months; this was enough to confirm the principle and allowed us to expand DryLab to include “DryLab-GC”. The software worked just as we had hoped, and we put it out to 4 different labs who were each willing to evaluate it. In each case the response was very positive, as summarized in an LCGC paper at that time. One example for a pesticide mixture is shown below (a) with a resolution map vs. heating rate (b). (a) (b) In this case, there are several peak reversals as the heating rate is changed, and the best separations occur at intermediate heating rates. Similar results for other samples surprised each of our evaluators, who expected the best separation at the lowest heating rate … or possibly at the highest rate. Despite this encouraging start for DryLab-GC, sales of the product were never close to break-even. We were HPLC people, and never got into the mindset of the GC crowd (or they never got into ours). MOSES. Expert systems for chromatography were a hot item beginning in the late 1980s, and we submitted our second proposal to NIH in 1992 for the development of an expert system for HPLC method development (Modular Optimization Software-Expert System, or MOSES). The proposal was approved (possibly because of the acronym), and we eventually spent about three years on the project with total funding of over $500,000. Work toward a final piece of software started well, but eventually it became clear that what we were trying to do was going to be very complicated, and require lots more time and money. So it ended up as an expensive, but useful, learning experience. Similar

38

efforts in other labs turned out about the same; in some cases commercial software resulted, but none of it ever achieved any significant impact. Several years later we were somewhat more successful in simply integrating DryLab into an automatic method development system that Waters marketed for about a year. Similar approaches have been picked up by other companies since 2005, but with, I believe, limited use so far. I tried to put all of this into focus in a brief 2012 article for LCGC. “2-D” DryLab. Our next big project was the expansion of DryLab for the simultaneous optimization of two different conditions that can change selectivity. In 1991 the original DryLab for optimizing %B or gradient time had been expanded for similar changes in pH, temperature, and other variables, but still one-variable-at-a-time. However, in 1992 we entered into a small collaboration with Bill Hancock (then at Genentech) for the application of DryLab to peptide separations (with gradient optimization). On the basis of this initial work, Bill asked whether changes in temperature might also be worth looking at. So his group carried out some experiments which indeed showed some striking (and unexpected) changes in peak spacing when either temperature or gradient time was changed: rhGH peptide digest Four experiments with both gradient time and temperature varied then allow the optimization of both variables: 45oC in 45 min (peaks 7-15) entire chromatogram

This was exciting stuff at the time. On the basis of these results, we applied in 1995 for a third NIH grant to develop and evaluate software for carrying out this approach for any kind of sample. We speculated at the time that the simultaneous optimization of temperature and gradient time might be

60-min, 20oC 60-min, 60oC

18 20 22 24 26 28 30Time (min)

7-15

39

especially attractive, because only 4 experiments were required and changes in both variables could be made easily and automatically by the LC system … very convenient and potentially useful for “automatic method development”. The proposal was eventually funded for 2+ years and about $800,000, and we ended up with a very useful product. During this time we carried out systematic studies on a wide range of samples, where Peng Ling measured retention times as a function of both gradient time and temperature. When converted to changes in selectivity (α) as a function of change in these two variables, we were excited to see frequent large changes in α (20-30% average) for reasonable changes in temperature and gradient time. So the approach looked quite promising. Peng Ling’s work ended up in four articles for the Journal of Chromatography. We next contacted several labs that were engaged in method development and worked with them to see how this approach might help for several “real” samples. A couple of publications summarized our results for 14 different samples from several labs, with consistently encouraging results; one gradient elution example is shown below. (a) herbicides resolution map (b) predicted optimum (c) actual separation

separation

Today this approach to LC method development is favored in many labs … an interesting sequel to our 1980 modest (but unproductive) attempt at computer predictions (p. 26) of separation as a function of temperature and %B (which is equivalent to gradient time). The software also allowed the simultaneous optimization of any two conditions that affect selectivity. An example is shown below for the optimized separation of a mixture of 17 substituted anilines and benzoic acids by varying both gradient time and pH. x x x x

Optimized separation: pH 3.97, tG = 30 min

Predicted (Rs = 1.4

Actual (Rs = 1.3

°c

tG

40

The hydrophobic-subtraction model. Our final NIH grant received $800,000 in total funding, and was for a project to better understand and use column selectivity. This (eventual) 15-year project is discussed later in more detail (p. 44). 50+ Years with the Journal of Chromatography My first two (single author) papers were submitted to the Journal in 1960, after the Journal of Physical Chemistry had rejected them. One JPC reviewer commented that our findings had been well known for 50 years. When the papers were then sent to the Journal of Chromatography, the editor Michael Lederer wrote a very friendly letter, mainly pointing out some additional references that I might include (as well I might!). The rejection by JPC was fortunate, as it was the wrong journal to reach working chromatographers – even if more “prestigious”. In 1970 I was invited to join the editorial board of the Journal, then in 1986 Michael Lederer agreed to retire as editor, a position he had held since 1956. I was approached by the Journal to see if I was willing to become part of a 5-member team that would work with Michael for his last two years, then take over as joint editors of the Journal. I accepted and remained an editor until 2000, at which time John Dorsey took my place, and I reverted to the editorial board of the Journal. Before I retired as editor, papers sent to the Journal were directed to editors with relevant experience and competence in a given area. My area was the theory of liquid chromatography, which meant that a lot of theory papers were sent my way. My impression in 1987 (when I started as an editor) was that some of the theory papers then appearing in the Journal were not very strong, and often lacked experimental verification … so I made an effort to tighten standards in this area. I eventually interacted with several thousand authors during my 13 years as editor and really enjoyed the process; of course, not all my interactions with authors were harmonious. I was especially helped as an editor by two outstanding members of the Journal editorial board: Hans Poppe and Georges Guiochon. They were each an editor’s dream: superbly competent reviews and sent in promptly without need for reminders! The Journal also encouraged the production of special volumes, two of which I initiated and edited: Computer-assisted Chromatographic Method Development (volume 485 [1989]) and Retention in Reversed-phase HPLC (volume 656 [1993]). Joe Glajch helped edit the first volume, while Pete Carr and Dan Martire were co-editors for the second volume. On my retirement from the Journal as an editor, a retirement party/symposium was organized with most of the participants shown below. This was a memorable occasion for me, as well as the first of several subsequent “retirement” parties. In 2009 I was appointed a member of the Honorary Editorial Board

41

of the Journal of Chromatography. While this position no longer requires me to review papers for the journal, as of 2015 I continue to do so by choice.

The Netherlands (June 13–15, 2001), on the occasion of my retirement from the Journal

Top row: P. Schoenmakers, J. Dolan, P. Carr, L. Snyder, J. Kirkland, I. Molnar, K. Bij, J. Dorsey, R. Giese, R. Marx, S. Terabe, N. Tanaka; middle row: E. Heftmann, D. McCalley, G. Vigh, R. Kaliszan, Cs. Horváth, S. Poole, S. Rutan, E. Soczewinski; bottom row: P. Jandera, C. Poole, H. Poppe, V. Davankov.

Sale of LC Resources During the period 1998-2002 our laboratory business had grown substantially, and the lab – which had most of the employees – was potentially our main profit center. This growth was the result of our increased experience with pharmaceutical HPLC; we were now doing a lot of method development business with several large companies. But we found this business to be increasingly competitive, with larger lab-service companies having more resources (and in some cases better trained staff). So we began to be concerned about the future viability of our company, and then explored its possible sale. The end result was the successful sale of the entire company in 2002. Rheodyne bought the software and training parts of the business, and BioAnalytical Systems (BAS) took the laboratory. Three years later, Rheodyne lost interest in short courses, at which time

Me

Hans

Vadim

Hans

Erich

Peter John Pete Me Jack Imre Klass

John Roger

Rob

Shigeru Nobuo

Edward Sarah Sandra Csaba

Roman Gyula David

Pavel

Colin

42

John, Tom and I bought back that part of the business. Rheodyne had earlier lost interest in DryLab and sold it to Imre Molnar, who has been responsible for the product to the present time. Short courses. Speaking of HPLC teaching, any initial success I enjoyed can be attributed to content, rather than delivery. My teaching style has always been on the “dry” side, which proved acceptable to early audiences composed largely of PhD level students, but less so when less experienced students began to predominate. On one occasion (~1988) I became ill just before a planned visit to Lilly. As I was unable to make the trip, I asked my contact at Lilly whether Tom Jupille could substitute for me. They reluctantly accepted this substitution. A few months later, when Lilly wanted another inhouse course, they phoned Tom and asked “whether Dr. Snyder would mind if Tom continued to teach instead of me”. I was happy with this change, as was Tom … and presumably the students at Lilly. After 1990, Tom and Derek Southern handled most of the short course teaching. Derek, played an important role in LC Resources after joining the

company in the late 1980s; he quickly became a respected advisor and much more than just an employee. Derek had previously been involved with the manufacture of HPLC columns at several companies, starting with Shandon in the early 1970s. In addition to his extensive HPLC experience, he was a “natural” as a short course teacher. For all practical purposes, Derek eventually became a fourth principal of the company. When LC Resources

was reconvened in 2005, John and Tom took over the teaching; John has been increasingly involved with teaching in other countries. Speaking of short courses, the ACS course that Jack and I started teaching in 1971 was joined after1990 by Joe Glajch and John Dolan. Joe and John then continued the course by themselves after 1995, until the course was discontinued around 2000. A get together in 2003 after a symposium honoring Jack’s Career at PittCon

John Jack Lloyd Joe Glajch

Derek Southern

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The Hydrophobic-subtraction (H-S) Model In looking around for new worlds to conquer in the late 1990s, I was reminded of some impressions picked up when in 1982 I first started consulting with different clients. At that time a frequent expedient during method development was a change of column in order to change selectivity, an approach that was time consuming and often unsuccessful. Also common then was an unwanted variation of selectivity from one batch of a particular column to another (e.g., Zorbax C18). So it appeared that there was a need for a better understanding of column selectivity, and some means for the easy application of this understanding to the latter two problems; i.e., so as to enable a quicker selection of a column of the right selectivity. In 1998, John Dolan and I organized an exploratory meeting with Jack Kirkland, Pete Carr, John Dorsey, and Uwe Neue, from which a column-selectivity research plan emerged. We then submitted a proposal to NIH that aimed at a more accurate procedure for characterizing the selectivity of reversed-phase columns. Before experimental work began, I had been playing with some data that Pete had published in 1996: values of k for about 80 solutes and several different C18 columns. Application of the solvation equation model to these data by Pete had yielded predictions that were accurate to about ±15%. I then plotted data for one column vs. another, and determined the average deviation (error) for each solute from the regression line. I was surprised to see that the latter errors were much smaller than those from solvation-equation predictions; i.e., better accuracy for a 1-parameter vs. a 5-parameter model! We later found that steric hindrance is much more significant in the stationary phase than in solution, hence requiring different solute parameters than those used for solvation-equation predictions. A year or so later, we began our NIH-sponsored experimental program, using 87 test-solutes and 10 different columns. Nan Wilson at LC Resources oversaw this work and brought it to a successful conclusion. Log-log retention plots for one column vs. another were carried out (a), and the residuals (δlog k) examined (b). x x x x

44

(a) (b)

(expansion of [a])

For many of the solutes, deviations δ log k in (b) were found to exceed twice the experimental error , meaning that these deviations were definitely “real”, not artifacts. Our final results would have been less easily interpreted, had we not achieved a remarkable ±0.5% repeatability in k during the time data were collected. It was next found that values of δlog k for certain solutes of related structure were highly correlated (r2 > 0.9), suggesting that they resulted primarily from just one solute-column interaction (other than hydrophobic). An example of one such correlating group, later associated with steric interaction, is show below. This then led to a general equation for retention as a function of solute (η’, σ’, α, β, κ) and column (H, S*, A, B, C) parameters:

log k = log kref + η’H - σ’S* +α’B + β’A + κ’C The quantity kref is for a reference compound (ethylbenzene) and the individual terms of the equation refer to specific solute-column interactions: hydrophobic (η’H), steric (σ’S), hydrogen bonding of an acidic (α’B ) or basic (β’A) solute, and electrostatic interaction (κ’C). The equation predicted values of k with an accuracy of ±1%. These results were complemented by later more detailed studies which established the H-S model (and the

log k (column-2)

log k (column-2)

δ log k

log k (column-1)

δ log k

log k (column-1)

r2 = 0.99

δ log k - δ log k plot avg. r2 = 0.97

45

above equation) as a fundamental description of column selectivity. The column parameters (H, S*, etc.) are of primary practical importance, as they can be used to select columns of similar or different selectivity. By the end of 2003, NIH support for this column-selectivity project ended, but work continued until the end of 2015 with the help of volunteers. Ongoing lab support was provided by Dan Marchand at the University of Wisconsin, Chris Heard at our old lab (now BAS) under the direction of John Dolan, and several collaborative studies. A few years later the US Pharmacopeia offered an online database which allowed different columns to be compared in terms of column selectivity by means of our model. Each column in the database required prior testing to evaluate its selectivity characteristics (H, S*, etc.). This column testing was continued at BAS until 2012, at which time the work was taken over at Gustavus Adolphus College by Dwight Stoll, a former student of Pete Carr. As of 2015, there are about 700 different columns in the database. Dan Marchand Dan Marchand had worked for LC Resources since 1990 and left

for a professorship at the University of Wisconsin in 2001. From 2004 until the end of 2011, Dan carried out work in support of the H-S model in his lab (initially with the help of Ken Croes). Dan had always wanted to teach at the college level, and since joining UW has done an outstanding job, honored by at least one award for distinguished achievements during his tenure at the university. He has been a good friend for over 20 years.

We also enjoyed the continuous support of Uwe Neue (Waters) as a consultant to the project, until his untimely death in 2011. In our final review of the H-S model in 2012 for a chapter in volume 50 of Advances in Chromatography, we noted that

Uwe Neue The authors wish to dedicate this chapter to the memory of

Dr. Uwe Neue (deceased), an outstanding scientist who made numerous contributions to the field of chromatography during his unfortunately short lifetime. Uwe was part of a small group who contributed to the initial planning of research on what eventually became the hydrophobic-subtraction model. Over the next dozen years, he offered many important suggestions that helped guide the project to a successful conclusion, as well as coauthored two recent publications [50,51]. His review of this chapter was carried out during his final days, which to us represents a remarkable example of his great dedication and sense of responsibility. He will be missed.

Important support to the project was also supplied from 2002 to 2005 by the Product Quality Research Institute (PQRI), an organization supported by various companies, universities and federal regulatory bodies who had an interest in pharmaceutical research. Once a large enough number of columns had been characterized in terms of selectivity, collaborative (multi-lab) studies were organized with the help of PQRI that confirmed several important features of the H-S model:

46

• highly reproducible measurement of the H-S column parameters (H, S*, A, B, C) • an ability to select columns of equivalent selectivity • an ability to select columns of very different selectivity (“orthogonal” columns)

Loren Wrisley (then at Wyeth, now at Pfizer) was especially helpful as a participant in these PQRI collaborations. In our study of equivalent selectivity, he provided an outstanding example of the accuracy of the H-S model, which employs a column comparison function Fs that measured differences in the selectivity of two columns. An equivalent column is needed, when it is necessary to replace a used column in an HPLC assay, and the original column part number is no longer available (or its selectivity varies from batch to batch). An equivalent column requires a small value of Fs. As seen below, the starting column in (a) (with Fs defined as 0) could be matched with equivalent columns (b) and (c) where Fs < 5. However column (d) with a larger value of Fs is no longer equivalent (two peaks marked by the arrow have merged together). A dozen other examples from this collaboration further confirmed the ability of the database to select equivalent columns. Later a similar collaboration was arranged to demonstrate the ability of a column of very different selectivity to be selected, in order to detect “hidden” peaks; i.e., a new sample component that was not present in the sample used during method development, and which overlaps one of the original peaks. Again, Loren provided a good example of the ability of the column database to select a column of very different selectivity. The original method provided the separation of (a), (a) where a new compound (#6) was present in this later sample, but is overlapped by peak (#3) and therefore “hidden”.

(a)

(b)

(c)

(d)

Luna C18(2) (original column) Fs = 0 Prodigy ODS (3), Fs = 1 Inertsil ODS-3, Fs = 2 J’Sphere H80, Fs = 10

Original method 1 2

4 5

3 + 6

47

With the orthogonal separation (b), the new peak (b) is well separated from other peaks. This allowed Wyeth to learn that a new impurity (compound-6) was present in some production samples. Fortunately for Wyeth, this only occurred for samples that had no t been released from production. Other laboratories in this collaboration reported similar results (but did not show the presence of new impurities in their samples). The H-S model has been further developed in recent years, confirming its fundamental nature and therefore its applicability to a broad range of questions related to column selectivity. The nature of the individual solute-column interactions has now been detailed to the point where a clear picture exists of their essence. In the process, two previously unknown interactions have been fully characterized: steric interaction (S*), which is quite different from the seemingly related column property shape selectivity, and column hydrogen-bond basicity (B) which arises from pairs of adjacent vicinal silanols. The success of our approach to column selectivity has resulted from the use of a model based on the principles of physical-organic chemistry, unlike other column comparisons which emphasize chemometrics.

Pete Carr Pete Carr played a major role in the development of the

hydrophobic-subtraction model, as well as worked with us earlier on the solvent-selectivity triangle and the above-mentioned special issue of the Journal of Chromatography (p. 41). Pete and I developed a close relationship over the years based on our many shared interests; I have probably exchanged more emails with Pete over the past decade than anyone else I know. I could say a lot about Pete, much of it summarized in the May 2013 issue of LCGC at the time Pete received the Lifetime Achievement in Chromatography Award.

Development of the H-S Model: a summary.

• I became aware that a change of column is often used to achieve either equivalent or different selectivity, but only by trial-and-error (1982)

• data in a Pete Carr paper suggest a theoretical approach for characterizing column selectivity (1997)

• meeting held to discuss a research plan and subsequent SBIR proposal (1998) • initial data lead to cross-plots of log k for different columns, with measurement of

deviations due to non-hydrophobic interactions (2000) • the initial H-S model is completed and published (2002) • the characterization of selectivity for the initial 10 C18 columns is expanded to

several hundred columns of different type (2005)

1

2

6

3

5

4

“Orthogonal” method

48

• with support from PQRI, USP creates a column selectivity website; it eventually comprises results for about 700 columns

• collaborative studies are completed with a dozen or so PQRI laboratories to confirm the accuracy of the model and its use for selecting similar or different columns (2006)

• studies of a more fundamental nature are carried out to better understand column selectivity in terms of the H-S model (2011-2015); this work confirmed and described two previously unknown interactions that contribute to column selectivity (so-called steric interaction and column hydrogen-bond basicity)

• more than a decade of research is summarized in a chapter in volume 50 of Advances in Chromatography (2012)

From the early 2000s until the present, the H-S model has been a personal obsession (but without any financial return). Introduction to Modern Liquid Chromatography The 3rd edition of this work, which appeared in late 2009 (with a publication date of 2010), had three personal aims for the authors (Jack, John and myself):

• update the successful 2nd edition (so-called “Bible” of HPLC), which sold 22,000 copies

• summarize and organize what we had learned for posterity

• make some money

The first two goals were achieved, and the book was widely praised … but sadly this book had sold only about 3000 copies by 2015. I leave the reader to speculate on reasons for its disappointing sales. Taking stock In late 2011 my final retirement from chromatography began, and was essentially complete by 2015. From the foregoing account, the reader can appreciate that the overall direction of my career was largely unplanned. Instead I bounced from place to place and project to project – largely as a result of fortuitous events and associates. The overall story resembles the life of the movie protagonist Forrest Gump. Like Gump I was unexpectedly fortunate, sometimes oblivious to the flow of events, and in some ways not very bright. I did however have some advantages: my exposure in graduate school to physical-organic chemistry and model building, and my early introduction to chromatography. I was also favored by an early start on my future career, at age nine.

In The Snoring Bird by Bernd Heinrich, the author commented on a book by his father Gerd Heinrich, Burmesiche Ichneuminae (Burmese ichneumons or wasps), Parts I and II. His father had served in the German army in World Wars I and II, and in 1945 became a

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refugee with his family. The two books were found many years later by his son, in storage in an old barn.

To the undiscerning eye, these two books would have no more meaning than they did to the chickens that had scratched among their pages. Yet I knew what they had meant to Papa. … He understood all too well the fleeting nature of existence. Somehow, against all odds, he had not only survived, escaped and started with nothing all over again. But he had gone on to produce this work that would long outlive him. And here it was, covered in chicken shit, scattered in the dust and unread. I thought, How immortal is a life work if the subject matter is so obscure that hardly a soul takes an interest in it?

Our work differed from Heinrich‘s in being perhaps more ephemeral than obscure, but the end result (“… hardly a soul takes an interest in it” ) may likely prove similar. And what is meant by the title of this biography (“An Accidental Career in Science”)? There is a second quote from The Snoring Bird:

So many of the stories Papa had told to me about his life had seemed too full of coincidences and strokes of fabulous luck to be believable.

And so was my career the beneficiary of numerous coincidences and strokes of luck (not all described here), if not involving the many life-and-death episodes experienced by “Papa” during WWII. But we are all the product of unanticipated events that shape our lives … some more so than others … some more fortunate than others. In looking back at my career, a central theme has been uncovering the principles of liquid chromatography, followed by their application and dissemination in articles, short courses and books, culminating in the 3rd edition of Introduction to Modern Liquid Chromatography. These various activities were personally enjoyable, and no doubt useful at the time. For the most part, however, they appear self-limiting; further work along these lines seems unlikely to greatly improve the practice of HPLC. Or at least it appears so to me . . . it was a good time to conclude my own career.

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Selected Publications Adsorption chromatography “Linear Elution Adsorption Chromatography. II. Compound Separability with Alumina as

Adsorbent”, L. R. Snyder, J. Chromatogr., 6 (1961) 22 Principles of Adsorption Chromatography. The Separation of Nonionic Organic Compounds, L.

R. Snyder, Marcel Dekker, New York, 1968 "Solvent Selectivity in Adsorption Chromatography on Alumina. Nondonor Solvents and Solutes",

L. R. Snyder, J. Chromatogr., 63 (1971) 15 "The Mechanism of Solute Retention in Liquid-solid Chromatography and the Role of the Mobile Phase in Affecting Separation. Competition vs 'Sorption'", L. R. Snyder and H. Poppe, J. Chromatogr., 184 (1980) 363 "Practical Optimization of Solvent Selectivity in Liquid-solid Chromatography Using a Mixture-design Statistical Technique", J. L. Glajch, J. J. Kirkland and L. R. Snyder, J. Chromatogr., 238 (1982) 269 “Mobile Phase Effects in Liquid-solid Chromatography", L. R. Snyder, in High-performance Liquid Chromatography: Advances and Perspectives, Vol. 3. Cs. Horváth, ed., Academic Press, New York, 1983, p. 157

“Solvent Selectivity in Normal-phase TLC”, L. R. Snyder, J. Planar Chromatogr. 21 (2008) 315 “Localization in Adsorption Chromatography”, L. R. Snyder, J. Planar Chromatogr. 25 (2012) 184

Petroleum analysis

“Applications of Linear Elution Adsorption Chromatography to the Separation and Analysis of Petroleum. III. Routine Determination of Certain Sulfur Types”, L. R. Snyder, Anal. Chem., 33 (1961) 1538 “Distribution of Benzcarbazole Isomers in Petroleum as Evidence for their Biogenic Origin”, L. R. Snyder, Nature, 205 (1965) 277

"Qualitative Analysis of Petroleum Compound Types by Linear Elution Adsorption Chromatography", L. R. Snyder, Anal. Chem., 38 (1966) 1319 "Petroleum Nitrogen and Oxygen Compounds", L. R. Snyder, Accts. Chem. Res., 3 (1970) 290

Gradient elution “Linear Elution Adsorption Chromatography. VII. Gradient Elution Theory.”, L. R. Snyder , J.

Chromatogr., 13 (1964) 415. "Optimized Solvent Programming for Separations of Complex Samples by Liquid-solid

Adsorption Chromatography in Columns", L. R. Snyder and D. L. Saunders, J. Chromatogr. Sci., 7 (1969) 195

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"Gradient Elution in High Performance Liquid Chromatography. I. Theoretical Basis for Reversed-phase Systems", L. R. Snyder, J. W. Dolan and J. R. Gant, J. Chromatogr., 165 (1979) 3

"Gradient Elution", L. R. Snyder, in High-performance Liquid Chromatography. Advances and

Perspectives, Vol. 1, Cs. Horváth, ed., Academic Press, New York, 1980, Chap. 4 "Measurement and Use of Retention Data from High Performance Gradient Elution. I. ", M. A.

Quarry, R. L. Grob and L. R. Snyder, J. Chromatogr., 285 (1984) 1 "Prediction of Precise Isocratic Retention Data from Two or More Gradient Elution Runs. An

Analysis of Some Associated Errors", M. A. Quarry, R. L. Grob and L. R. Snyder, Anal. Chem., 58 (1986) 907

"HPLC Separations of Large Molecules. A General Model", L. R. Snyder and M. A. Stadalius, in

High-performance Liquid Chromatography. Advances and Perspectives, Vol. 4, Cs. Horváth, ed., Academic Press, New York, 1986, p. 195

"Conventional Chromatographic Theory Versus ‘Critical’ Solution Behavior in the Separation of

Large Molecules by Gradient Elution", M. A. Stadalius, M. A. Quarry, T. H. Mourey and L. R. Snyder, J. Chromatogr., 358 (1986) 17

"The Linear-solvent-strength Model of Gradient Elution", L. R. Snyder and J. W. Dolan, Adv.

Chromatogr., 38 (1998) p. 115 “Peak Compression in Reversed-phase Gradient Elution”, U. D. Neue, D. H. Marchand and L. R.

Snyder, J. Chromatogr. A, 1111 (2006) 32 High-performance Gradient Elution, L. R. Snyder and J. W. Dolan, Wiley-Interscience, Hoboken,

NJ, , 2007 HPLC Method development and computer simulation

"Classification of the Solvent Properties of Common Liquids", L. R. Snyder, J. Chromatogr., 92 (1974) 223

"Computer Simulation as a Means of Developing an Optimized Reversed-phase Gradient-elution Separation", J. W. Dolan, L. R. Snyder and M. A. Quarry, Chromatographia, 24 (1987) 261

"The Design of Optimized HPLC Gradients for the Separation of either Small or Large Molecules.

II. Background and Theory", B. F. D. Ghrist and L. R. Snyder, J. Chromatogr., 459 (1989) 25 "Temperature as a Variable in Reversed-phase HPLC Separations of Peptide and Protein Samples. I. Optimizing the separation of a growth hormone tryptic digest", W. Hancock, R. C. Chloupek, J. J. Kirkland and L. R. Snyder, J. Chromatogr. A, 686 (1994) 31 "Combined Use of Temperature and Solvent Strength in Reversed-phase Gradient Elution. I. Predicting Separation as a Function of Temperature and Gradient Conditions", P. L. Zhu, L. R. Snyder, J. W. Dolan, N. M. Djordjevic, D. W. Hill, L. C. Sander and T. J. Waeghe, J. Chromatogr. A, 756 (1996) 21

Practical HPLC Method Development, 2nd ed., L. R. Snyder, J. J. Kirkland and J. L. Glajch, Wiley-Interscience, New York, 1997 “Simultaneous Variation of Temperature and Gradient Steepness for reversed-phase HPLC method development. I. Application to 14 different samples using computer simulation", J. W.

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Dolan, L. R. Snyder, N, M. Djordjevic, D. W. Hill, D. L. Saunders, L. Van Heukelem and T. J. Waeghe, J. Chromatogr. A, 803 (1998) 1 “Introduction to Modern Liquid Chromatography”, 3rd edn., L. R. Snyder, J. J. Kirkland and J. W. Dolan, Wiley, Hoboken, NJ, 2010 “Optimizing Selectivity During Reversed-phase HPLC Method Development: Prioritizing Experimental Conditions”, L. R. Snyder and J. D. Dolan, J. Chromatogr. A, 1302 (2013) 45

Prep LC "A Simplified Description of HPLC Separation under Overload Conditions, Based on the

Craig-distribution Model. II. Effect of Isotherm-type, and Experimental Verification of Computer Simulations for a Single Band", J. E. Eble, R. L. Grob, P. E. Antle and L. R. Snyder, J. Chromatogr., 384 (1987) 45

"A Simplified Description of HPLC Separation Under Overload Conditions. A Synthesis of Two

Recent Approaches", L. R. Snyder, G. B. Cox and P. E. Antle, Chromatographia, 24 (1987) 82

"A Simplified Description of HPLC Separation Under Overload Conditions, Based on the Craig-distribution Model. V. Gradient Elution Separation", J. E. Eble, R. L. Grob, P. E. Antle and L. R. Snyder, J. Chromatogr., 405 (1987) 51

"Preparative HPLC under Isocratic Conditions. III. The Consequences of Two Adjacent Bands Having Unequal Column Capacities (ws)", G. B. Cox and L. R. Snyder, J. Chromatogr., 483 (1989) 95

Column selectivity

“Column Selectivity in Reversed-phase Liquid Chromatography. I. A General Quantitative Relationship”, N. S. Wilson, M. D. Nelson, J. W. Dolan, L. R. Snyder, R. G. Wolcott and P. W. Carr, J. Chromatogr. A, 961 (2002) 171 “The hydrophobic-subtraction model of reversed-phase column selectivity”, L.R. Snyder, J.W. Dolan, P.W. Carr, , J. Chromatogr. A, 1060 (2004) 77 “Choosing an Equivalent Replacement Column for a Reversed-phase HPLC Assay Procedure”, J. W. Dolan, A. Maule, D. Bingley, L. Wrisley, C. C. Chan, M. Angod, C. Lunte, R. Krisko, J. M. Winston, B. Homeier, D. V. McCalley, L. R. Snyder, J. Chromatogr. A, 1057 (2004) 59. “Orthogonal Separations for Reversed-phase Liquid Chromatography”, J. Pellett, P. Lukulay, Y. Mao, W. Bowen, R. Reed, M. Ma, R.C. Munger, J. W. Dolan, L. Wrisley, K. Medwid, N. P. Toltl, C. C. Chan, M. Skibic, K. Biswas, K. A. Wells and L. R. Snyder, J. Chromatogr. A, 1101 (2006) 122 “Contributions to Reversed-phase Column Selectivity. I. Steric Interaction”, P. W. Carr, J. W. Dolan, U. D. Neue, L. R. Snyder J. Chromatogr. A, 1218 (2011) 1724 “Contributions to Reversed-phase Column Selectivity. II. Cation Exchange”, D. H. Marchand, P. W. Carr, D. V. McCalley, U. D. Neue, J. W. Dolan, and L. R. Snyder J. Chromatogr. A, 1218 (2011) 7110 “The Hydrophobic-subtraction Model of Reversed-phase Column Selectivity”, L. R. Snyder, J. W. Dolan, D. H. Marchand, P. W. Carr, Adv. Chromatogr. 50 (2012) 297.

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“Contributions to Reversed-phase Column Selectivity. III. Column Hydrogen-bond basicity”, P. W. Carr. J. W. Dolan, J. G. Dorsey, J. J. Kirkland and L. R. Snyder, J. Chromatogr. A, 1395 (2015) 57

Miscellaneous

“Two Stage Gas-Liquid Chromatography”, M. C. Simmons and L. R. Snyder, Anal. Chem., 30 (1958) 32 "The Surface Structure of Porous Silicas", L. R. Snyder and J. W. Ward, J. Phys. Chem., 70 (1966) 3941

"An Experimental Study of Column Efficiency in Liquid-solid Adsorption Chromatography.

HETP Values as a Function of Separation Conditions", L. R. Snyder, Anal. Chem., 39 (1967) 698 "Rapid Separations by Liquid-solid Chromatography. Qualitative and Quantitative Analysis of Hydrogenated Quinoline Mixtures", L. R. Snyder, J. Chromatogr. Sci., 7 (1969) 595 "Dispersion in Segmented Flow through Glass Tubing in Continuous-flow Analysis: The Nonideal

Model", H. J. Adler and L. R. Snyder, Anal. Chem., 48 (1976) 1022 "Coated-open-tubular Chromatography with Flow Segmentation. II. Experimental Study and

Optimization for Size-exclusion Separation", J. W. Dolan and L. R. Snyder, J. Chromatogr., 185 (1979) 57

"Boxcar Chromatography: A New Approach to Increased Analysis Rate and Very Large Plate

Numbers", L. R. Snyder, J. W. Dolan and Sj. van der Wal, J. Chromatogr., 203 (1981) 3 "Separation of Macromolecules by Reversed-phase High Performance Liquid Chromatography.

Pore-size and Surface-area Effects for Polystyrene Samples of Varying Molecular Weight", J. P. Larmann, J. J. DeStefano, A. P. Goldberg, R. W. Stout, L. R. Snyder and M. A. Stadalius, J. Chromatogr., 255 (1983) 163

"Column-efficiency in High-performance Liquid Chromatography as a Function of Particle

Composition and Geometry, and Solute Capacity Factors k'", R. W. Stout, J. J. DeStefano and L. R. Snyder, J. Chromatogr., 282 (1983) 263

"A Model of Protein Conformation in the Reversed-phase Separation of Interleukin-2 Muteins",

M. Kunitani, D. Johnson and L. Snyder, J. Chromatogr., 371 (1986) 313 "Some Practical Applications of Computer Simulation for GC Method Development", G. N.

Abbay, E. F. Barry, S. Leepipatpiboon, T. Ramsted, M. C. Roman, R. W. Siergiej, L. R. Snyder and W. Winniford, LC.GC Mag., 9 (1991) 100