In previous columns, we have discussed the development of the first gel-permeation chromatograph (1) and the work by Csaba Horváth at Yale University (New Haven, Connecticut) in the laboratory of Professor S.R. Lipsky, leading to the first modern, high-pressure liquid chromatograph (2). However, although these instruments usually are called the first liquid chromatography (LC) instruments, they actually were preceded by another complex instrument that was based upon the principles of LC on ion-exchange resins: the automated amino acid analyzer developed in 1958 at The Rockefeller Institute for Medical Research (New York) by S. Moore, W.H. Stein, and D.H. Spackman (3,4).

The amino acid analyzer opened an entirely new field for research, permitting the elucidation of the composition of proteins. We might even say that the rapid expansion of biochemistry would have been impossible without it. However, because the majority of chromatographers are not in the biochemical field, this development is relatively unknown to the general public. In this installment, we try to fill this gap in the history of chromatography.

To understand the importance of this development, a brief summary of proteins and amino acids, and the development of separation based upon ion-exchange, is useful.

Amino acids are the building blocks of proteins: very complex, mostly α-helical, three-dimensional polymers consisting of hundreds of amino acids connected by peptide bonds. Proteins are the key gene-expressed compounds in life processes, and this is reflected by their name: "protein" comes from the Greek word proteios, meaning "of primary importance." This term was first used in 1839 by the great Swedish chemist JÖrs Jakob Berzelius (1770–1848). The study of the amino acids has a long history, leading to the slow recognition of the individual amino acids. The 1902 Nobel laureate Emil Fischer (1852–1919) showed in the early 1900s how amino acids are bound to each other forming polypeptides, the building blocks of proteins (5).

In the first part of the twentieth century, research on proteins and amino acids was carried out along two lines: as part of nutrition studies and investigating their chemistry and composition. Decades of painstaking work by many researchers established that the proteins of the humans and animals contain 20 amino acids, from which 10 cannot be synthesized by the human body, but must be taken nutritionally. The proper amount and balance of the essential amino acids in our food intake is very important. However, these 20 natural amino acids are only part of the amino acids occurring in nature: we know of the existence of hundreds, or even thousands of additional ones.

Naturally, researchers in biochemistry, nutrition, and other life sciences fields wanted to identify the individual amino acids present in proteins and determine their quantitative levels. For this, the proteins must first be hydrolyzed to break the peptide bonds. This usually is done by boiling the peptide with an acid for an extended time; it is a delicate operation, as some amino acids can be destroyed during hydrolysis, and various safeguards are used to prevent and compensate for it.

Various methods have been developed for the determination of the individual amino acids. Emil Fischer was the first to show that the esters of the amino acids can be distilled, and he used the fractional distillation of the esters prepared from protein hydrolysates to obtain a more precise understanding of their original composition. Other methods introduced in the first part of the twentieth century involved selective precipitation as insoluble salts, colorimetric analysis, and microbiological assays, but these were complicated and time-consuming operations (6). A.J.P. Martin and R.L.M. Synge first described partition chromatography in 1941 for column chromatography (7) and then its paper chromatography version — introduced in 1944 by Martin's group (8) — represented a major breakthrough, permitting separation and simultaneous determination of amino acids in a straightforward manner. Martin and Synge received the 1952 Chemistry Nobel Prize for the invention of partition chromatography.