History of Liquid chromatography


Brief History of Liquid chromatography

What follows are brush strokes of the historical development of liquid chromatography and chromatography that were taken from articles published by renowned HPLC researchers. It is not intended to be an exhaustive or complete description of the development of HPLC and is surely unfair to many researchers whose contribution was fundamental to this essential technique in virtually all modern sciences, but reading it may help to understand the mechanisms, and perhaps to improve current processes. Many of the events cited may briefly show the origin and some milestones that allowed us to get here(11-17).

The beginning cannot exclude the invention of HPLC, which we owe to Mikhail Semyonovich Tsweet who developed it in 1900. Tsweet was born in 1872 in Asti, Italy, the son of an Italian mother and a Russian officer. At a young age he moved to Switzerland where he graduated and took up botany. He then followed his father when he was called back to Russia and settled in St. Petersburg. He had to revalidate his degrees in Moscow, where he pursued academic activities, as well as in Warsaw and Lithuania. He first used the name chromatography in his publications in 1906 (see Figure 1.8). In his work he filled glass tubes with a solid, in the case of the figure with calcium carbonate and equilibrated it with a liquid, carbon disulfide. After introducing chlorophylls in solution at the upper end, he eluted the liquid through the column, whereupon the pigments separated in the column, revealing a sequence of colored bands. At that time he created the name chromatography, from the Greek chroma (color) and grapho (writing), that is, writing in color.

Figure 1.8. Drawings of the article published in 1906 by Tsweet. It includes in (a) an apparatus with five simultaneous columns with a mercury manometer and a rubber bulb for dispensing the mobile phase. It also shows in (b) a funnel-type column 2-3 mm in diameter and 20-30 cm in length mounted on a Kitasate and in (c) a chromatographic separation of pigments, using carbon carbon carbonate as the stationary phase and carbon disulfide as the mobile phase.Reproduced with permission from LC-GC.

In reality, the sample components were not eluted from the column as we do today, but formed a color pattern within the column representing the composition of the chlorophylls. The method had great repercussion, but naturally it had great limitations as it could not visualize colorless components, and did not allow the elution of the components of the column.

Thin layer chromatography (TLC) presented a solution to this problem. TLC could be considered as an open column chromatography, so that by accessing the material, specific or universal development could be carried out as required. For example, the application of a ninhydrin spray followed by heat reveals the colorless amino acids, which turn blue. Iodine vapors, when the dry plate is exposed to its vapors after the run, fix on organic compounds with double or triple bonds, giving them a reddish-brown color. And the simple spraying of sulfuric acid followed by heating in an oven at 100°℃ carbonizes the organic compounds which are visualized as dark spots on the white plate.

HPTLC of chamomile oil, different disclosures

A) UV 254 nm with and without filter

B) UV 366 nm Post derivatization

C) Anisaldehyde drifting Reflactance R+T Transm.

Figure 1.9.Thin plate HPLC of chamomile oil showing the result of the use of various developers for the visualization of the separated compounds.

One of the first precursors of this technique was Edgar Lederer and it had more recent advances with Egon Stahl. The impact of TLC was immediate, and due to its simplicity and low cost it quickly gained ground in the field of analytical chemistry. At the time of the wider diffusion of liquid and gas HPLC, TLC continued to develop in Eastern Europe. It is still used today, although with much less diffusion due to the advance of HPLC.

Shortly after, liquid HPLC (LC) had a very significant breakthrough in the 1940s when Archer J.P. Martin and Richard L.M. Synge created partition HPLC or liquid-liquid HPLC (LLC), as opposed to the liquid-solid HPLC (LSC) that predominated at that time, and such was the impact of this development that they received the 1952 Nobel Prize in Chemistry for it.

Martin was doing research on vitamin E at Dunn Nutritional Laboratory, and was experimenting with the separation of carotenes heterogeneous phases. He had constructed for this purpose a system of 45 chained tubes that served as decanting ampoules, separated by 90 valves that prevented the return of the liquid to the previous ampoule. The scheme of his apparatus was not published.

Synge on his part investigated the amino acids of wool in IWS, and the method employed consisted of acetylating the amino acids to extract them in chloroform. He noted with enthusiasm the difference in the chloroform-water partition coefficients of the acetylated amino acids, which presented promising prospects for liquid-liquid extraction, and for this he was contacted by Martin, who was very experienced in countercurrent distribution and had developed an apparatus that had made him well known in Cambridge. Shortly afterwards they found themselves working at the Wool Research Association in Leeds, UK, where they had to create another apparatus because the one they had was not compatible with the water-chloroform system. The new continuous flow apparatus had 39 "theoretical plates" (Figure 1.10 represents a system devised by Lyman Craig in the '40s (18) not necessarily identical to Martin's system, but showing a countercurrent extraction train used in those years).

Figure 1.10. Countercurrent distribution train developed by Lyman Craig in 1944, which greatly simplified the multiple extraction process in decanting ampoules. Published with permission of the American Chemical Society(18).


It was reportedly a complicated system, which had to be attended by two operators in 4-hour cycles, lulled to sleep by chloroform vapors. The results were not very satisfactory, and Martin and Synge tried to improve the system by creating variants. An intermediate system was a glass tube with the wool and cotton fibers oriented in the direction of the tube, with the chloroform moving in one direction and the water in the opposite direction. Neither worked as expected until finally Martin thought that it was not necessary to move the two liquids, that one could be left stationary in a column and move the other.

To achieve this purpose they impregnated the silica gel they used as a desiccant in the balance with water and with this material they packed a glass column, added the acetylated amino acid mixture and poured chloroform into the inlet, perhaps as in the schematic in Figure 1.11). The addition of methyl orange allowed them to follow the path of the amino acids through the column. It may sound very simple today, but it was certainly hard work to properly prepare the stationary phase (water) on a support that had not been fabricated for that purpose (silica gel) and to properly prepare the mobile phase (chloroform) in a reproducible way. Martin developed in parallel the theory of HPLC based on distillation concepts, and the paper was submitted in two parts in November 1941 to the Biochemical Journal, and published in December 1941 as A New Form of HPLC Employing Two Liquid Phases (19-20).

Figure 1.11. Schematic of the co-column of partition HPLC or LLC. The solid particle behaves as an inert support, and the stationary phase is the liquid that imbibes it, which is immiscible with the mobile phase and with the sample solvent.

Shortly thereafter Synge left the group to investigate amino acids in antibiotics, and was the first to elucidate the sequence in a polypeptide using paper HPLC.

Martin joined the Medical Research Council in 1948 and became involved in the analysis of fatty acids by HPLC. As his system had a polar stationary phase and a poorly polar mobile phase (water and chloroform), the partition coefficients did not favor it, so he decided to develop a hydrophobic stationary phase to retain the fatty acids. He achieved this with Howard (20) by treating kieselguhr (diatomaceous earth) with dimethyl dichlorosilane (see Figure 1.12).

Unlike the liquid-liquid partition chromatography developed in '41 where the attachment of the liquid to the stationary phase was a simple mechanical retention in the pores and therefore unstable, this reaction bound the silanizing molecule to the support by covalent bonding, leaving the nonpolar liquid (dimethylsilane) as the hydrophobic stationary phase, immiscible with the polar solvent of the mobile phase. Martin used this system to separate lauric acid from stearic acid by partitioning.

Figure 1.12. Silanization reaction by which Martin and Howard succeeded in covalently bonding a liquid, dimethyl dichlorosilane to the inert LLC support, kieselguhr or diatomaceous earth.

On the other hand, Martin had predicted the use of a gaseous mobile phase in 1941, but did not develop the idea until 1950 when he brought the silanized stationary phase to GC, publishing the method as gas-liquid partition HPLC in October 1950. This method proved its usefulness in a short time and was the basis for the development of gas-liquid HPLC (GLC).

Within a short time this material migrated back to liquid HPLC and was first used in 1967 by Aue and Hasting at Dalhouise University in Nova Scotia, but probably the first commercialized filler material using reversed phase was developed in 1972 by Kirkland at Dupont. This material was called Permaphase ODS, was of the pellicular type and consisted of 35 um diameter microspheres with a thin stationary layer of silica gel attached to octadecylsilane, totaling a spherical particle of about 50 um diameter. Prior to this, in 1966-1967 Csaba Horvath noted that the working pressure needed was greater than 1000 psi, and thinking that this technology deserved a new name he was the first to use the four letters we know today, HPLC (21).

In 1972, Majors developed at Varian the microparticle material of 10 μm silica gel bonded to ODS. This material was called MicroPak-CH. Shortly thereafter, in 1973 Csaba Horvath baptized the method as Reverse Phase.

In retrospect, the relatively limited diffusion of liquid-liquid partition HPLC may be striking today, although we can see that important causes for this were due to the quality of the support, not yet designed for these purposes, the difficulty in keeping the stationary phase stable on the solid support and the preparation of the mobile phase. Probably for this reason IUPAC published in 1972 an article stating that "the reversed phase is only a technique of historical interest"; however, the covalent bonding of a liquid to the supporting solid was another fundamental fact that led to the development of modern stationary phases, having the physical characteristics of a solid, but containing an interface liquid with stable bonding.

We can say that in the early 1970s HPLC was born, with the development of columns filled with efficient macroparticles and systems that allowed the elution of the mobile phase by the use of pumps and high pressure. As indicated above, the first particles used were of the pellicular type, consisting of small glass spheres of about 35 μm coated with a thin layer of silica gel covalently bonded to octadecylsilane (ODS or C18), resulting in a particle of about 50 μm.

Shortly thereafter, fully porous macroparticles of 10 μm in diameter were developed, and the supply of stationary phases of different functionality was diversified, but this will be developed in more detail in the following pages.

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21.A.J.P.Martin and Howard,Biochem.J.46,532(1950)-LL partition paraffin oil and n-octane on diatomaceous earth.

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