From Tswett’s “Farbschreibung” to modern Chromatography.
A historical overview
Almost every lecture and publication on this topic begins with the work of Michail Semjonowitsch Tswett . In his experiment he filled a test tube with Inulin and then poured a chlorophyll extract in ligroin over it. Then he added more ligroin. Initially the eluting liquid was colorless but then a yellow ring (carotene) appeared below a green ring at the top of the column, which, during the course of the experiment, separated clearly into green and yellow.
This experiment is often referred to as the birth of “modern” Chromatography. However, Leslie Ettre’s book “Chapters in the Evolution of Chromatography”, describes in comprehensive detail how there were “pioneers” in the field of chromatography well before Tswett and that their experiments are documented in the bible .
From Invention to Implementation
But how did things develop after Tswett published his findings? For a long time it was an uphill struggle for Tswett to get his technique recognized in expert circles. We can often see similar behavior today when a new method or alternative detection process is developed. Initially scepticism dominates and there is a search for arguments as to why a use will not be found for a certain method. As time goes by a good idea inevitably does assert itself. This is how it was, a couple of decades later, when Martin and Synge were awarded the Nobel Prize for their partition chromatography in 1952 [3-6]. However it took another decade for the foundation stone of modern chromatography to be laid. Jim waters was the first to develop a chromatograph for gel permeation chromatography. From this point on, HPLC found its way into many laboratories that were working on questions of the time, regarding the separation and purification of compounds.
The bells rang out for HPLC for the first time in 1973 when the first HPLC Symposium was held in beautiful Interlaken . The synthesis of new stationary phases and the high reproducibility of separating media played a large role in establishing the technology in important branches such as the chemical-pharmaceutical industry.
It is common knowledge that a lot of effort is needed before a technology can be used in an industrial process. Even today reversed phases based on silica gel are highly appreciated. Besides the high reproducibility of the manufacturing process, the very good mechanical and chemical stability of these materials have especially contributed to HPLC’s fast promotion to being the standard analytical process. On the other hand, alternative separating materials such as porous graphitized carbon or zirconium dioxide coated with polybutadiene lead a shadowy existence to this day, although the extended pH- and temperature stabilities of these stationary phases offer many advantages.
Smaller, faster, better.
Against this backdrop, there was no real innovation up to the beginning of the new millennium. Typical standard detectors such as UV- and fluorescence detectors were widely distributed and could be easily coupled to an HPLC system. The volume of a classic HPLC system was relatively high compared to today’s latest technology. This was also the reason why miniaturized HPLC processes played no role in the practical application. A perfect example is the work carried out by Tsuda and Novotny in 1978 . The injection stream had to be divided in order to avoid overloading the capillary columns. On the detection side a supplemental stream had to be set up. This compensated for the negative influence of the high volume in the detector cell but lowered the sensitivity. Therefore, even before the discussion of the results, the authors of the study come to the conclusion that it is just basic research for evaluating the separating efficiency of capillary columns. For more comprehensive experiments and thus also a routine application, it would be necessary to have better injection systems and detectors with lower cell volumes. Although the theoretical basis of chromatography and therefore also the advantages of miniaturized separation systems were already well documented when the first appliances were established, it took another four decades from the introduction of the first commercially available HPLC-system until a new milestone was reached. It was the positively ground breaking work of the team around Jorgenson that paved the way for this. In a publication from 1999 the authors describe an experimental set up for using fused-silka-capillaries with an internal diameter of 33µm packed with non-porous octadecyl-modified silica gel particles that had a diameter of 1.0µm . The system was operated under a pressure of 5,000 bar, whereby the injection pump described in the manuscript had a maximum pressure of 9,000 bar. To this day, this team’s experiments, which are described in diverse publications, stand out and illustrate that there is still great potential for further improvement in chromatographic systems.
MS, UHPLC and 1-2-3D.
The commercial availability of particles with a diameter of < 2 µm (so called, sub 2 µm particles) combined with an HPLC-system with a pump that could generate 1,000 bar and whose additional system components could withstand this pressure led to a minor “revolution” in the field of analytical separation techniques. At the time, the introduction of the UHPLC (Ultra high-performance liquid chromatography) only resulted in the shaking of heads and tired smiles in expert circles. Nowadays almost all companies that supply HPLC systems have pumps in their portfolio that can extend their pressure range to 1,000 bar. Interestingly, the call for better chromatographic resolution was accompanied by a development in the area of mass spectrometry, which in part, presented a competing technology to chromatography. With the introduction of the first mass spectrometer to routine laboratories, the prognosis was that the complex chromatography techniques would soon be a thing of the past, because, it was argued: a mass spectrometer can also separate co-eluting compounds. This is true! However, as time went by, it became clear that a mass spectrometer would also reach its limits rather quickly – particularly at that moment when the number of compounds simultaneously inserted in the ion source and the difference in their concentration are very high. It became clear very quickly that even one dimensional chromatography processes combined with mass spectrometry would not offer sufficient separating efficiency if, for example, a digested protein was to be analysed. Two dimensional separating techniques were suddenly being discussed enthusiastically at scientific conferences. Even though they were far removed from the idea of a routine application. Nowadays, in 2016, several companies offer two dimensional HPLC-systems but under no circumstances will there be stagnation in the development. Scientists in Peter Schoenmakers group are working on the development of three dimensional separating structures on a chip . This may seem extremely futuristic for many users, but very soon the vast technological advances will overcome the obstacles that currently exist. The sceptics only need to look at the rapid development that we have seen in the fields of telecommunication and computer technology since the beginning of the new millennium.
But what role does Micro-LC have in this? Although there are many areas with potential, the technique is currently only being used to a limited extent. What's more, the system volumes are so low in between times, that columns with an internal diameter of 300µm display a comparable separating efficiency to columns with an internal diameter of between 2.1mm and 4.6mm . In the fields of proteome research and bio-analytics, a process based on Nano-LC is used if the available sample quantities are limited. In other fields of application, where the available sample quantities are sufficient, classic HPLC or UHPLC systems are used. On the basis of the validation of an accredited method in our own lab, we have demonstrated that Micro-LC presents a real alternative here. In addition, more and more manufacturers are offering products for the fields of Micro- and Nano-LC. The necessary conditions for implementing Micro-LC are favourable and cannot be simply reduced to costs savings in the organic solvents – a topic that we will be taking a closer look at in the upcoming articles.
Do you have questions about Micro-LC or multi-dimensional separation? The team of experts at IUTA will be happy to answer them under: firstname.lastname@example.org
Authors: Thorsten Teutenberg, IUTA; Terence Hetzel, IUTA; Juri Leonhardt, IUTA; Denise Löker
Dr. Thorsten Teutenberg
Institut für Energie- und Umwelttechnik e. V. (IUTA)
 M.S. Tswet, 1903.
 L.S. Ettre, J.V. Hinshaw, Chapters in the Evolution of Chromatography, Imperial College Press, 2008.
 A.J.P. Martin, R.L.M. Synge, Biochemical Journal 35 (1941) 1358. DOI:10.1042/bj0351358
 A.J.P. Martin, R.L.M. Synge, Biochemical Journal 35 (1941) 91.
 A.J.P. Martin, Nobel Lectures - Chemistry 1942-1962, Elsevier, Amsterdam, 1952, p. 372.
 R.L.M. Synge, Nobel Lectures - Chemistry 1942-1962, Elsevier, Amsterdam, 1952, p. 372.
 W. Gehrke , Bayer Chromatography-A Century of Discovery 1900-2000.The Bridge to The Sciences/Technology, Elsevier Science B.V., Amsterdam, 2001.
 Tsuda, Novotny, Analytical Chemistry 50 (1978) 271.
 J.E. MacNair, K.D. Patel, J.W. Jorgenson, Analytical Chemistry 71 (1999) 700.
 B. Wouters, E. Davydova, S. Wouters, G. Vivo-Truyols, P.J. Schoenmakers, S. Eeltink, Lab on a Chip 15 (2015) 4415.
 T. Hetzel, D. Loeker, T. Teutenberg, T.C. Schmidt, Journal of Separation Science (2016).