Editors' Column
Liquid Chromatography of Proteins and Peptides The recent development of a series of new silica-based liquid chromato graphic (LC) stationary phases has created a virtual revolution in the ap plicability of LC to the separation of proteins and peptides. The new, col umn materials feature particles that are uniformly sized, mechanically sta ble, and fully porous. The pore size distributions are more uniform than ever before. And pore dimensions are larger than ever before to accommo date the very high molecular weight proteins and peptides under intensive study today. As molecular weight ascends, diffu sion coefficients and column efficien cies descend. Large molecules simply take longer to diffuse through the pores in fully porous stationary phases, leading to band broadening. To compensate, chromatographers have been moving to smaller and smaller diameter stationary phase particles. In classical techniques, par ticles were typically 100 μπι across. "Then the scientific community start ed to use 10-μηι supports," says Fred Régnier of Purdue. "This increased resolution enormously. Now we're at the five-μιη level, and we're experi menting with three-μπι supports." LC separations depend on just a few fundamental molecular properties. Molecules have size and shape, they have charge, and they exhibit varying degrees of hydrophobicity. Size exclu sion (SE) is used to separate molecules on the basis of size and shape. In ion exchange (IE) they are separated by charge. And reversed phase (RP) is used to separate compounds on the basis of hydrophobicity. Seldom are the distinctions so pure in real life, however. All three chromatographic modes have some size exclusion com ponent when fully porous particles are used. And SE is seldom a simple mat ter of molecules diffusing through pores without any chemical and physi cal interactions whatsoever. The situa tion is usually considerably more com plicated than that, involving what are
referred to as secondary interactions. These interactions are currently a topic of intense study and debate. The International Symposium on HPLC of Proteins and Peptides, held Nov. 16-17 in Washington, D.C., brought together hundreds of scien tists interested in the separation of biopolymers. Chairing the symposium were Fred Régnier of Purdue, Milton Hearn of St. Vincent's School of Medical Research in Australia, and C. Timothy Wehr of Varian Associates, Inc. The symposium was also sponsored by Varian.
Ion Exchange Modern IE is still strikingly similar to its classical counterpart in terms of the eluents that are used. But new packing materials have made it possible to perform faster separations at higher resolution than ever before. Proteins can be separated in 10-40 min with modern IE chromatography. This is 60 times as fast as classical IE, and the resolution one can achieve in 20 min is superior to what used to be achieved in 24 h with the classical columns, according to Fred Régnier. In addition, in most cases better than 90% of the enzymatic activity in the original sample is preserved. Thus, IE is an excellent preparative technique for enzymes. According to Régnier, who admittedly has been a proponent of IE for years, the technique will be catching on even more in the future, as the new packing materials become more and more widely available commercially.
Reversed Phase Literally hundreds of papers have appeared since the early seventies on the application of RP to peptide separations. The same columns used for peptides were not, however, applicable to proteins. Only in the last year or two have RP columns for proteins become commercially available, and the technical papers are just now starting to appear on the use of these columns
for biomolecular separations. The pore sizes of the new R P packings are larger than before, making it possible to separate larger molecules than ever before. Compared to IE, RP is often easier to use, and the columns are more efficient with R P , according to Stanley Stein of Hoffmann-La Roche, who presented the plenary lecture on RP of proteins and peptides. One must turn to IE, however, when the solvents used in RP would tend to denature the proteins under study. Chromatography of proteins and peptides with RP typically involves gradient elution, starting with a buffered aqueous solution and continuing with increasing proportions of propanol or perhaps acetonitrile, whereas only salt and water are used as eluents in IE. Neither salt nor water damages a molecule very much. There are, however, whole classes of proteins that are not denatured in the typical RP eluents. Interferon survives very nicely, Stein points out. Pituitary hormones like prolactin have been successfully chromatographed by RP. And there are proteins, such as the membrane proteins and some of the immune regulators, that cannot be separated at all without recourse to organic eluents. Stein has applied RP to the separation of the opiod peptides, beta endorphin, and the enkephalins. Beta endorphin is being used clinically in Japan as an analgesic for killing pain in childbirth, and the enkephalins are involved in the pain centers of the brain. Human leukocyte and fibroblast interferons have also been isolated by RP in amounts sufficient for structural studies. Stein is currently working on the traditionally difficult separation of some of the different kinds of collagen proteins found in the body.
Size Exclusion Proteins can now be separated across a wide range of molecular
ANALYTICAL CHEMISTRY, VOL. 54, NO. 1, JANUARY 1982 · 21 A
Editors' Column
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22 A · ANALYTICAL CHEMISTRY, VOL. 54, NO. 1, JANUARY 1982
